Sample processing barcoding bead compositions, methods, preparations, and systems

Abstract

An embodiment of a composition for target material separation, the composition comprising: a body and one or more molecules coupled to the body and configured for functionalization of the composition. In embodiments, each of the one or more molecules can comprise one or more of: a linker region; a polymerase chain reaction (PCR) segment or oligo binding region; one or more barcode regions; a unique molecular identifier; a preparation assistance segment; an activity segment; and a molecular scissors or cleavage region, wherein the various regions can be coupled together so as to provide functionality to the composition. The invention also comprises preparation of the composition and applications of various uses in the context of target material capture (e.g., from single cells or other biological material).

Description

[0001] Cross-reference to related applications This application claims the benefit of U.S. Provisional Application No. 62 / 945,006, filed December 6, 2019, which is incorporated herein by reference in its entirety. Technical Field

[0002] This invention relates generally to the field of cell capture and cell processing, and more specifically to novel and useful systems, methods and compositions for sample processing barcode beads for target material reactions.

[0003] background With increasing interest in cell-specific drug testing, diagnostics, and other assays, systems and methods that allow for the isolation, identification, and recovery of single cells have become highly desirable. Single-cell capture systems and methods have shown particular advantage for these applications. However, the associated processes and protocols for single-cell capture and subsequent analysis often must be performed in a specific manner and with high precision to properly preserve the cells. Furthermore, efficiently recovering target material from high-density platforms presents numerous challenges. Moreover, the composition of materials can be significantly modified for applications involving the capture and recovery of target material in a manner that allows for single-cell analysis. Therefore, these processes can be time-consuming for users, potentially requiring extensive and iterative manual library preparation and selection processes, may not be suitable for automation, and may therefore lead to cell damage (e.g., in terms of undesirable loss of viability), high background noise rates, increased false positive rates, or other unreliable experimental results.

[0004] Therefore, there is a need in the field of cell capture and cell processing to create new and useful systems and methods for sample handling and target material recovery, minimizing the steps required in the preparation of libraries of target biomaterials. Some of these embodiments utilize molecular barcoding (e.g., by using barcoded oligonucleotides in a workflow, typically delivered to a reaction environment including functional particles). There is also a need to create methods for streamlining the preparation of large quantities of the described barcoded bead embodiments. Brief description of the attached diagram Figure 1 A schematic diagram depicts an embodiment of the composition used for the reaction of the target material.

[0006] Figure 2 A schematic diagram depicts an alternative embodiment of the composition for reacting with the target material.

[0007] Figure 3 A schematic diagram depicts an embodiment of a connector molecule contained in a composition for reacting a target material.

[0008] Figures 4A-4CVariations of compositions that can be used to capture mRNA into cDNA synthesis reactions or protein tagging interactions are described.

[0009] Figure 5 Variations of compositions incorporating components that simplify library preparation procedures are described.

[0010] Figure 6A Variations of compositions that can be used to capture mRNA into cDNA synthesis reactions are described.

[0011] Figure 6B Variations of compositions that can be used in protein tagging reactions are described.

[0012] Figure 6C-6E Variations of compositions incorporating thermally unstable joint elements are described.

[0013] Figure 7 Variations of compositions incorporating molecular scissor regions are described.

[0014] Figure 8A-8M The variations in the coupling between functionalized molecules and substrates are described.

[0015] Figure 9A and Figure 9B Variations of the compositions that can be used in ATAC-seq operations are described.

[0016] Figure 9C-9E Variations of compositions containing restriction sites are described.

[0017] Figure 10 A flowchart illustrating an implementation scheme for the ATAC-seq method is provided.

[0018] Figure 11 A flowchart depicts a method for preparing the composition.

[0019] Figure 12A A flowchart depicts variations of the method for preparing composition particles.

[0020] Figure 12B and Figure 12C Variations of the steps used to prepare the composition are described.

[0021] Figure 13 The variations in the synthesis of oligonucleotide molecules are described.

[0022] Figure 14 The variations in the synthesis of a portion of an oligonucleotide molecule are described.

[0023] Figure 15The detailed steps of synthesizing a portion of an oligonucleotide molecule are described.

[0024] Figures 16A-16E The synthesis of a group of oligonucleotide molecules with unique barcodes coupled to particles was described.

[0025] Figures 17A-17B Alternative variations in the synthesis of a group of oligonucleotide molecules with unique barcodes coupled to particles are described.

[0026] Description of preferred implementation scheme The following description of preferred embodiments of the present invention is not intended to limit the invention to these preferred embodiments, but rather to enable any person skilled in the art to make and use the invention.

[0027] 1. Benefits The invention described can provide several advantages over conventional systems, methods and compositions.

[0028] This invention provides the following benefits: it offers compositions for facilitating the capture, extraction, and / or recovery of target biological materials from samples, while providing a barcode for each biomarker molecule recovered from a sample partition, which may be a discrete single cell within the sample. Such compositions may contain materials that have been modified from their natural state (e.g., in providing structural differences from natural compositions). Furthermore, this invention relates to combinations of materials that are non-natural (e.g., the described and claimed compositions have no naturally occurring counterparts).

[0029] The present invention also includes novel compositions and component chemistry of the substrate to simplify the library preparation process.

[0030] The present invention also includes novel compositions having cleavable sites that allow for the separation of target materials, and having the ability to monitor cleavage and / or quantify components processed from biological samples.

[0031] This invention also provides the benefit of offering a mechanism for the efficient recovery of target materials (e.g., beads, cells, released nucleic acid materials, etc.) from the high aspect ratio pores of a high-density capture platform. Recovery is typically difficult and inefficient in this configuration due to the densely packed pores of the capture platform. The described recovery mechanism also subjectes the target material to acceptable amounts of shear and other potential stresses that would otherwise hinder downstream processing steps.

[0032] The present invention also provides the following benefits: providing a method for preparing beads for capturing target molecules and / or coupling molecules to a substrate (e.g., chamber wall), wherein the molecule contains a set of detectable unique barcodes for sample processing.

[0033] The present invention also provides the benefit of reducing the burden on system operators associated with the process of recovering target material from the borehole, where standard processes may be inefficient / labor-intensive.

[0034] This invention also offers the benefit of improved efficiency in recovering target material (without recovering non-target material). Therefore, selective recovery efficiency can reduce downstream costs associated with processing reagents and other materials (due to reduced required volume), processing burden, and improved signal-to-noise ratio. For example, this invention allows system operators to purchase smaller volumes of reagents, reduce the number of separations required for successful amplification of target molecules, and avoid the need for SPRI-based washing and size selection of target oligonucleotide products to separate them from other oligonucleotide tags that do not contain the product but are left over from one processing step to the next. Such improved target product recovery and reduced non-target product residue can also reduce the complexity of data analysis and provide more usable data related to the desired biomarker analysis. This can result in cost savings, reduced reagent waste, or obtaining any other suitable results.

[0035] Because the described compositions, methods, and systems provide a greater number of target reads, the present invention also provides the benefit of providing greater sequencing depth for the desired target.

[0036] This invention also provides the benefit of enabling at least partial automation of processes involving single-cell capture, target material recovery, and subsequent processing. For example, human operators can be eliminated from some or all of the methods. Furthermore, the system and / or method can achieve better accuracy in terms of process performance compared to conventional systems and methods. Some of these inventions are also better suited for the full automation of liquid handling robots.

[0037] Alternatively or concurrently, the present invention may provide any other suitable benefits.

[0038] 2. Functional bead composition like Figure 1As shown, an embodiment of composition 100 for target material separation comprises: a body 110 and one or more molecules 120 coupled to the body 110 and configured for functionalization of composition 100. In an embodiment, each of the one or more molecules 120 may include one or more of the following: a linker region 130; a polymerase chain reaction (PCR) segment or oligonucleotide binding region 140; one or more barcode regions 150; a unique molecular identifier 160; a preparation-facilitating segment 170; an active region 180; and a molecular scissors or cleavage region 190, wherein the various regions may be coupled together (e.g., sequentially) to provide functionality to the composition. In application, composition 100 may be provided as a set of functionalized particles, each functionalized particle having a set of coupled oligonucleotide molecules for various assays, said oligonucleotide molecules being configured to assist extraction operations, amplification processes, size-based purification processes, binding processes, release and recovery processes, and other reactions (e.g., molecular reactions) for single-cell analysis.

[0039] Composition 100 can be configured to operate with a system configured to perform single-cell analysis in manual, semi-automatic, and / or automatic operating modes. Embodiments, variations, and examples of such systems are described in one or more of the following: U.S. Application No. 13 / 557,510, entitled “Cell Capture System and Method of Use,” filed July 25, 2012; U.S. Application No. 14 / 289,155, entitled “System and Method for Isolating and Analyzing Cells,” filed May 28, 2014; U.S. Application No. 15 / 422,222, entitled “System and Method for Isolating and Analyzing Cells,” filed February 24, 2017; U.S. Application No. 15 / 815,532, entitled “System and Method for Retrieving and Analyzing Particles,” filed November 16, 2017; and U.S. Application No. 15 / 815,532, entitled “System and Method for Isolating and Analyzing…” Cells” and U.S. Application No. 16 / 115,059, filed August 28, 2018; each of the applications is incorporated herein by reference in its entirety.

[0040] Composition 100 can be configured for use in processes and reactions associated with one or more of the following: reverse transcription (RT-reaction), immunochemistry, DNA reaction, mRNA FISH reaction, proximity ligation reaction, bridging amplification reaction, catalytic enzymatic reaction, hybridization reaction, restriction digestion reaction, amplification reaction (e.g., mRNA and / or DNA PCR), and other suitable reactions. Such reactions can be performed on-chip and / or off-chip, with embodiments, variations, and examples of microfluidic chips for single-cell analysis described in the following: U.S. Application No. 13 / 557,510, entitled “Cell Capture System and Method of Use,” filed July 25, 2012; U.S. Application No. 14 / 289,155, entitled “System and Method for Isolating and Analyzing Cells,” filed May 28, 2014; U.S. Application No. 15 / 422,222, entitled “System and Method for Isolating and Analyzing Cells,” filed February 24, 2017; and U.S. Application No. 15 / 815,532, entitled “System and Method for Retrieving and Analyzing Particles,” filed November 16, 2017; each of these applications is incorporated herein by reference in its entirety.

[0041] 2.1 Functional bead core The role of the host 110 is to provide a substrate, to which one or more molecules 120 can be coupled to provide functionalization for the composition in order to carry out the corresponding assays and reactions.

[0042] Regarding morphology, the body 110 may be in the form of a microsphere. Alternatively, the body 110 may be in the form of a non-spherical body (e.g., an ellipsoid, prism, polyhedron, amorphous, etc.), wherein the cross-section taken through the body 110 is non-circular. However, the body 110 may optionally have other suitable forms. Regarding dimensions, the body 110 may have a diameter (or feature width) of 5-50 micrometers, with tolerances of [missing information]. + The particle size ranges from 0.05 micrometers to 5 micrometers. Furthermore, the uniformity of the body 110 across the entire particle population allows for the achievement of desired recovery efficiency performance after each step of the intended single-cell process. In a specific example, the body 110 has a size of 20 micrometers. + The diameter is 1 micrometer; however, variations of the exemplary body 110 may have other forms.

[0043] In one embodiment, the body 110 has a feature size configured such that only a single body 110 of composition 100 can enter the pore of the chip along with a single target cell, for co-localization and co-capture of single-cell-particle pairs within a single pore. However, the body 110 of composition 100 may have other suitable feature sizes configured for other microfluidic or non-microfluidic assay applications.

[0044] Regarding density, body 110 is configured to have a density greater than that of the processing liquid intended for use with composition 100 (e.g., in relation to a specific reaction or assay), such that composition 100 settles in the processing liquid due to gravity during operation. In an embodiment, the density of body 110 is greater than 1.02 g / cm³. 3 However, in variations, the body 110 may have other suitable densities. For example, in some embodiments, the body 110 may be configured to have the same density as the intended processing liquid (e.g., to facilitate steps in which the processing liquid flow carries the body 110). In yet other embodiments, the body 110 may be configured to float relative to the processing liquid, such that the body 110 is floating and can be used to separate target or non-target materials from a sample.

[0045] Regarding density and morphology, the substrate 110 can be a continuous substrate (e.g., at the micrometer scale, at the nanometer scale, at the sub-nanometer scale). Optionally, its variations are as follows: Figure 2 As shown, body 110 may comprise clusters of smaller bodies 115 (e.g., having a morphology scaled down from the macroscopic morphology of body 110, or having other morphologies). Due to the surface aggregation of the smaller bodies 115, such a configuration can provide a larger total surface area, resulting in macroscopic properties of a single body for oligonucleotide synthesis (e.g., in terms of near-rigidity / other mechanical properties), and / or the ability to dissolve upon use in an assay (e.g., after capture) to provide the desired surface chemical properties. The clusters of smaller bodies 115 may be surrounded (e.g., encased therein) by cluster material 116, which may temporarily or “permanently” maintain the cluster morphology. In an example, cluster material 116 may comprise a hydrogel having properties suitable for the intended use of the composition (e.g., in terms of crosslinking, solubility, porosity, density, thermal properties, optical properties, charge, composition, mechanical properties, other physical properties, etc.). In relevant applications, the cluster material 116 can maintain the cluster morphology of the smaller bodies 115 during the use phase of the assay, and can then be dissolved or otherwise removed to transform the smaller bodies 115 into a non-clustered state (e.g., to provide improved proximity to the surface chemistry of each smaller body).

[0046] In one example, composite microspheres are made by surface reactions of multiple small microspheres (e.g., having a diameter of 0.5 micrometers) with larger microspheres (e.g., having a diameter of 19 micrometers), such that the composite microspheres have a total diameter of 20 micrometers. However, the surface area of ​​the composite particles is significantly enhanced due to the presence of smaller microspheres or certain reactive groups arranged in a specific pre-designed array.

[0047] In implementations, the substrate and surface properties can vary to provide significant performance flexibility. For example, larger microspheres may be rigid materials, while smaller microspheres may be hydrogels. In another example, larger microspheres may be nonmagnetic, while smaller microspheres may be magnetic. In yet another example, larger microspheres are magnetic, and smaller microspheres are magnetic or paramagnetic. In yet another example, larger microspheres may be made of transparent materials, while smaller microspheres may be optically (e.g., bright-field or fluorescent) encoded. In yet another example, larger microspheres may be prepared as soluble, while smaller microspheres are insoluble. Another embodiment of composite microspheres may comprise a set of base rigid microspheres coated with a thin (e.g., 1-3 micrometer layer) hydrogel or other material to provide increased reaction surface area. Such innovative microspheres will also provide the additional advantage of allowing biomarkers of certain sizes to permeate into the microspheres to partially participate in specific reactions. Another example of composite microspheres may comprise solid particles (e.g., 20 micrometers in diameter) having microtunnels (e.g., 0.1-2 micrometers in diameter) extending from the surface of the composite microsphere to its center. In some cases, these microtunnels may extend across the entire diameter of the particle. In yet another embodiment, the microtunnels are pores that increase the total surface area of ​​the composite material. In yet another embodiment, the large microspheres may have a thin coating on their surface that has a different functional composition compared to the internal composition. The top surface may be cross-linked, but the internal material may be soft or soluble.

[0048] In some variations, each smaller body 115 may be identical in properties and composition; however, in others, one or more smaller bodies 115 may be configured to have different properties, compositions, and distributions within the cluster (e.g., from the core to the surface) to provide different functions for different portions of the assay or reaction. For example, a first region of the cluster (e.g., the surface) may have a first set of properties, compositions, and / or surface chemistry for a first portion of the assay or reaction, may be dissolved or otherwise removed, and then a second region of the cluster (e.g., the core) may have a second set of properties, compositions, and / or surface chemistry for a second portion of the assay or reaction.

[0049] In a particular example, a group of approximately 750 smaller bodies 115 are aggregated in a soluble hydrogel to provide a total diameter of 20 micrometers, having a total surface area ~7.5 times that of a single continuous 20-micrometer particle. Each smaller body 115 comprises polystyrene (PS-DVB) crosslinked with divinylbenzene and has a diameter of 1 micrometer (with appropriate tolerances). In another example, body 110 may comprise a hydrogel, wherein the smaller bodies are composed of a polyacrylamide matrix, and the cluster material comprises a disulfide crosslinking agent (e.g., BAC). However, variations of the examples can be configured in other suitable ways.

[0050] Regarding thermal properties, body 110 is configured to operate between a lower temperature limit (e.g., related to low-temperature reactions and processes, related to storage, etc.) and an upper temperature limit (e.g., related to high-temperature reactions and processes). In a specific instance, the lower temperature limit is -20°C to 4°C (e.g., for refrigeration), and the upper temperature limit is 90°C to 120°C (e.g., for denaturation reactions). However, body 110 can be configured for other operating temperatures.

[0051] Regarding physical properties, body 110 is configured to maintain its structure in solution (e.g., in a buffer solution during storage, in a solution during assays). Therefore, body 110 is configured to be non-swellable and non-leaching. However, in alternative embodiments, body 110 may be configured to swell to the desired amount (e.g., to achieve the desired size or morphology for handling or use in application), to leach certain compounds (e.g., treatment reagents) for assays, and / or to dissolve in a desired manner during assays or other processes. In yet another embodiment, the particles may have a specifically tailored swelling property such that their use in particular buffer solutions and / or physical conditions allows the particles to readily enter micropores, but may be trapped within the micropores under certain buffer conditions. Furthermore, regarding physical properties, body 110 may be configured to have a desired degree of hydrophilicity (e.g., in the range from hydrophilic to hydrophobic) in relation to the conduct of assays or other processes. Regarding surface properties related to fluid contact, body 110 may be configured to have the desired wettability (e.g., in terms of contact angle, etc.). Therefore, variations of the body 110 can have suitable crosslinking types (e.g., chemical crosslinking, physical crosslinking, etc.) and crosslinking percentages (e.g., 1%-10% crosslinking of acrylamide, 30%-99% crosslinking of other materials, other suitable crosslinking ranges) to provide the required level of stability under conditions of use.

[0052] Regarding other surface properties, body 110 can be configured to have a desired porosity (e.g., 200-2000 Å, etc.). Body 110 can additionally or optionally be configured to have a desired loading density (LD) to enable an appropriate junction density (e.g., by providing attachment points on body 110 to provide a more robust detectable signal during use), wherein the addition of body 110 is described in more detail in Section 2.2 below. Furthermore, body 110 may contain surface groups (e.g., hydroxyl groups, amine groups, carboxyl groups, sulfide groups, silanol groups, etc.) for coupling the junction molecules described in Section 2.2 below. In examples, the desired loading density (LD) can be as low as 1 μmol / g or as high as several hundred μmol / g of functional group density.

[0053] Regarding magnetic properties, the body 110 can be configured to respond to a magnetic field (e.g., in determinations involving the separation and / or recovery of target or non-target materials). In variations of the body 110, certain regions of the body 110 (e.g., the core region) can be magnetic (e.g., magnetic, paramagnetic, etc.), and certain regions of the body 110 (e.g., the shell region) can be nonmagnetic. Regarding surface properties, the body 110 can be configured to be charged or uncharged to facilitate binding with target materials or to facilitate the preparation of molecules involving functionalization.

[0054] Regarding optical properties, the body 110 can be configured to be non-fluorescent (e.g., so as not to interfere with optical-based detection and measurement). However, in variations, the body 110 can be configured to be optically detectable (e.g., by non-fluorescent form, by fluorescent form, by infrared detection form, by thermal detection form, etc.), for example, for tracking purposes.

[0055] Regarding mechanical properties, the body 110 may be configured to have the desired hardness (e.g., measured according to a Mohs scale, or according to other hardness scales) in order to maintain the desired hardness level during the application. Additionally or optionally, the body 110 may be configured to have desired mechanical properties associated with one or more of the following: rigidity, elastic properties (e.g., in terms of modulus, in terms of plasticity and elastic deformation, etc.), viscoelastic properties, fatigue resistance, fracture resistance, shear strength, compressive strength, tensile strength, rheological properties (e.g., under wear conditions), and other mechanical properties.

[0056] Regarding composition, body 110 may comprise one or more of the following: polystyrene, polystyrene-divinylbenzene, polymethyl methacrylate (PMMA), silica, silicone, non-porous glass, porous glass, coated glass, agarose, acrylamide, polyacrylamide, iron, steel, or ceramic materials, and / or combinations of one or more suitable materials. As described above and below, different regions of body 110 may comprise different materials (e.g., the core region may comprise a first material, and the shell region may comprise a second material). In some embodiments, there may be more than one shell region or more regions in other configurations such as amorphous or ordered spatial arrangements.

[0057] Specific examples of body 110 include polyacrylamide (e.g., as described in more detail below), silica (e.g., silicone), polystyrene, or PMMA with a diameter of 15-25 micrometers (e.g., where the smaller diameter allows for slight swelling in a manner still suitable for use in microfluidic structures), a surface porosity of 80-1500 Å, a crosslinking rate of polymer beads between 20% and 80% (e.g., 60% polystyrene crosslinked by divinylbenzene or 80% polystyrene crosslinked by divinylbenzene), surface groups (e.g., amine groups, hydroxyl groups, silanol groups) for coupling chemical connectors (such as C18 tag connectors), and polyethylene glycol (PEG) functionalization for reaction efficiency. Variations of specific examples may have a magnetic (e.g., magnetic, paramagnetic) core or shell to allow for magnetic functionality (e.g., for separation and recovery).

[0058] 2.2 Functional Molecules like Figure 1As shown, composition 100 further comprises one or more molecules 120, which are coupled to body 110 and configured for functionalization of composition 100. In embodiments, each of the one or more molecules 120 may comprise one or more of the following: a linker region 130; a polymerase chain reaction (PCR) segment 140; a barcode region 150; a unique molecular identifier 160; a preparation aid region 170; an active region 180; and a molecular scissor region 190, wherein the various regions may be coupled together (e.g., sequentially) to provide functionality to the composition. One or more molecules 120 can serve to provide the desired chemistry (e.g., binding chemistry) for different reactions or processes, and in variations, the inclusion of specific oligonucleotides in one or more molecules can make one or more molecules suitable for mRNA binding, binding to CITE sequencing probes, oligonucleotide-labeled antibodies, oligonucleotide-labeled peptides, oligonucleotide-labeled lipids, oligonucleotide-labeled metabolites, modified genomic DNA, unmodified genomic DNA, DNA ATAC sequencing, Hi-C sequencing, cut-n-tag sequencing, bridging amplification, proximity ligation, other molecular reactions, other protein tagging operations, and / or other reactions. Furthermore, one or more molecules can be used for various sequencing platforms (e.g., next-generation sequencing platforms, Illumina...). TM Regions (e.g., specific adaptors, primers) of sequencing platforms are suitable for assisting library preparation operations. Therefore, one or more molecules 120 can simplify manual or automated steps associated with sequencing or other reactions by incorporating specific oligonucleotide segments.

[0059] In embodiments, one or more molecules 120 may comprise a single molecule, a set of identical molecules, or a set of different molecules (e.g., first and second molecules, or more than one different molecule) distributed throughout the body 110. For example, in a reaction involving mRNA capture and cDNA synthesis, one or more molecules 120 may comprise an oligonucleotide molecule having a first sequence for mRNA binding and a second sequence associated with the generation of a complementary cDNA strand. Similarly, in a reaction involving protein tag binding, one or more molecules may comprise a molecule having a first sequence and a molecule having a second sequence, the first sequence being used to detect antibody binding by detecting the tagging of the antibody by the oligonucleotide tag, and the second sequence being used for synthesis. In another embodiment, different sets of molecules for providing forward and reverse primers may be present in one or more molecules 120 to allow bridging amplification to amplify certain nucleic acid fragments from a single cell initially bound to the microspheres. The relative proportions of the various forward or reverse primers can be adjusted such that only certain sizes of cDNA are maximized during bridging amplification (e.g., products less than 600 base pairs or greater than 300 base pairs). However, the sequence of one or more molecules 120 can be adapted to other reactions and processes, variations of which are described below in conjunction with different structural features of one or more molecules 120. Binding groups may also be present in 120 in certain proportions so that enzymes are bound to the microspheres during enzymatic reactions, allowing these enzymes to process and produce reaction products of mRNA, ensuring the products reach only certain sizes or preventing the products from exceeding certain base sizes. Optionally, structural features may exclude certain enzymes (e.g., nucleases or restriction endonucleases) or other functional parts from being closely approximated to the body 110 in order to adjust the size of the retained molecule to the desired size (e.g., any molecule longer than 300 bp is digested to a smaller size).

[0060] 2.2.1 Molecular-Connector like Figure 1 As shown, the body 110 may include a set of connectors including connectors 130, wherein the connectors 130 serve to control the density and spacing of one or more molecules 120 coupled to the body 110 in a manner that provides a sufficient number of molecules / sites for the occurrence of a reaction. The set of connectors also serves to control the density and spacing of one or more molecules 120 in a manner that prevents molecular folding at the body surface or otherwise forms undesirable structures (e.g., secondary structures, tertiary structures, etc.), or in other embodiments, to control the density in a manner that promotes such structures.

[0061] In this implementation, the number of adapters in the set is configured to be greater than the number of target molecules per single cell targeted for the binding reaction. In one example, the number of target molecules per cell is on the order of 500,000 to 1,000,000 molecules or molecular fragments; therefore, in this example, the set of adapters may contain 10 7 -10 10 One connector for positioning 10 on each body 110. 7 -10 10 A set of full-length oligonucleotides, wherein an excess of full-length oligonucleotides results in the capture of more mRNA (or other molecules) during the reaction. However, in other embodiments, the set of adapters may contain an additional number of adapters.

[0062] In an embodiment, connector 130 includes a branching connector configured to provide a suitable density of oligonucleotide molecules on the surface of body 110 and to provide suitable spacing between adjacent oligonucleotide molecules. In variations, the branching connector is a dendritic macromolecule (e.g., a symmetrical dendritic macromolecule, an asymmetrical dendritic macromolecule, a doubler, a trebler, labeled, unlabeled, etc.) that provides branches with attachment nodes. In one variation, the dendritic macromolecule may be a Y-shaped dendritic macromolecule comprising a source node (e.g., for attachment to a region of body 110 or the proximal end of body 110) and two terminal nodes (e.g., for attachment to one or more functional oligonucleotide molecules of molecule 120 or for attachment to a subsequent dendritic macromolecule at the distal end of body 110). In a particular instance, the branching connector is a symmetrical double phosphoridamide dendritic macromolecule; however, variations of the particular instance may use other core chemistry (e.g., carbosilane, thiolation, etc.) and structure. Therefore, in other variations, dendritic macromolecules can have any other suitable number of attachment sites, chemistry and / or structure to provide spacing and coupling sites between the oligonucleotide molecule and the host 110.

[0063] Furthermore, branched adapters can be configured for selective attachment (e.g., having functional groups specific to a particular chemical substance) and / or selective cleavage (e.g., for releasing oligonucleotide segments during processing, such as molecular scissors).

[0064] like Figure 3As shown, dendritic macromolecules that can be used as linkers can be formed by coupling a set of base reagents to an initial branching center, starting from the initial branching center, and then adding several generations of base reagents sequentially until the desired dendritic macromolecule size and the number of terminal branches (e.g., the exponent of the generation) are reached. The type, generation, and molecular weight of the base reagent functional groups can produce a hydrodynamic diameter corresponding to a desired diameter corresponding to the oligonucleotide helix width (e.g., ~2 nm) in order to achieve the desired density of oligonucleotide molecules coupled to the host 110 through linker design. However, the final diameter (or other characteristic size) of the dendritic linker can be configured to match other design constraints or configured in other suitable ways.

[0065] 2.2.2 Molecular-PCR segment like Figure 1 As shown, each of the one or more molecules may also contain one or more polymerase chain reaction (PCR) segments 140, which are configured to perform PCR-related reactions (e.g., amplification). The PCR segments may contain PCR primers for performing the PCR reaction. As shown above regarding different types of nucleic acid-related reactions and protein-related reactions (and in... Figures 4A to 4C As shown in the diagram, PCR primers for different sequences of one or more molecules 120 may be the same as or different from each other. For example, in a first variation, a first portion of one or more molecules 120 may include a first PCR primer segment 141 associated with a first stage of the reaction (e.g., mRNA binding, antibody binding, binding to other protein tags, etc.), and a second portion of one or more molecules 120 may include a second PCR primer segment 142 associated with a second stage of the reaction (e.g., cDNA synthesis, other synthesis, other tagging, other binding, etc.).

[0066] In other variations, the PCR segment 140 may additionally or optionally include a PCR handle segment 143, which is detectable and configured for quality control of the composition. However, variations of one or more molecules 120 may additionally or optionally omit the PCR handle segment 143.

[0067] In one implementation, the PCR segment 140 is directly coupled to the terminal portion (or other portion) of one of the adapters 130 in a set of adapters. However, in other variations, the PCR segment may be coupled to other portions of the oligonucleotide molecule in other ways.

[0068] In the implementation, PCR segment 140 may have 5-30 bases and may contain custom or non-custom primers; however, in alternative variations, PCR segment 140 may have other suitable numbers of bases.

[0069] 2.2.3 Molecular-Barcode Region and Unique Molecular Identifier (UMI) like Figure 1 As shown, each of one or more molecules 120 may include a barcode region 150, which functions to uniquely identify biological material (e.g., cellular material) treated or derived (e.g., synthesized) using one or more molecules 120 of composition 100. The barcode region 150 may be configured to reduce noise associated with the detection signal and available reads (e.g., related to assigning sequencing reads to the correct barcode and reducing wasted reads). With respect to the preparation method 400 described in more detail below, the accuracy of the barcode regions 150 across all molecules coupled to a particular host 110 (involving minimizing unintentional deletions, substitutions, or additions) can thus result in a low false-positive error rate (e.g., matching of the signal to an incorrectly barcoded molecule).

[0070] like Figure 1 As shown, barcode region 150 may be coupled to PCR segment 140 (e.g., at the distal end of PCR segment 140 relative to body 110), or may optionally be coupled to other parts of one or more molecules 120.

[0071] Barcode region 150 may contain one or more barcode segments, the preparation and assembly of which will be described in more detail in Section 4 below. In some variations, barcode segments may include a portion for assembly (e.g., a handle, such as a connection handle or PCR extension handle), which may optionally be used as part of the barcode or independently of the barcode segment. In some variations, each barcode segment may be 2-20 nucleotides long; however, in alternative variations, each barcode segment may have other suitable lengths. Preferably, each barcode segment has a Hamming distance greater than 2 (e.g., the number of substitutions required to make two nucleic acid strings identical); however, in alternative variations, barcode segments may have other suitable Hamming distances. Furthermore, each barcode segment may be configured not to end with GG (or other sequences less suitable for a particular sequencing platform); however, barcode segments may be configured in other suitable ways. Barcode region 150 can be constructed from one or more segments to create 1 million to 100 million unique barcodes of suitable length; however, variations can produce other suitable numbers of unique barcodes. In a particular instance, the barcode segments are selected from a set of 875 (or more) 7-mers with a Hamming distance of 2, not terminated by GG bases, where the sequence is not naturally occurring. In a particular instance, the barcode region contains more than one segment, which, when assembled together, creates 50 million unique barcodes. However, variations in the specific instance can be configured in other suitable ways.

[0072] like Figure 1 As shown, each of one or more molecules 120 may contain a unique molecular identifier (UMI) 160, which serves as a molecular tag to allow a sequencing platform (e.g., a next-generation sequencing platform) to identify the input molecule being processed. Each of one or more molecules 120 may have a single UMI or more than one UMI. Furthermore, the UMI 160 may be coupled to a barcode region 150 (e.g., at the distal end of the barcode region 150), such as... Figure 1 As shown, or at other locations along one or more molecules 120.

[0073] 2.2.4 Molecular-Preparation Auxiliary Section like Figure 1 As shown, each of one or more molecules 120 may optionally include one or more preparation aid sections 170, which are used to simplify or otherwise reduce processing steps associated with certain operations.

[0074] In one variation, such as Figure 5As shown, the preparation auxiliary segment 170 can be configured to simplify library preparation steps by incorporating molecular sequences that would normally have to be performed in a separate step (e.g., originally manually). More specifically, one or more molecules 120 may contain a p5 linker (e.g., for Illumina...). TM The first preparation aid segment 170a associated with the flow cell, wherein, in some variations, the first preparation aid segment 170a contains a portion of the P5 linker and the sequence of the associated index. In variations, the first preparation aid segment 170a may be coupled to a barcode region 150 (e.g., adjacent to the body 110, or other suitable region). One or more molecules 120 may also contain a P7 linker (e.g., for Illumina...). TM The platform and configuration for cDNA synthesis include a second preparative helper segment 170b, which can be added during the same step, during reverse transcription, or in other separate steps. In some variations, the second preparative helper segment 170b contains the sequence of random primers configured to randomly bind to the target mRNA molecule closer to the 3' end of the mRNA molecule and prevent elongation of the 5' end of the mRNA molecule. Therefore, during reverse transcription, the cDNA strand terminates near the random primer segment. A ligase then ligates the random primers to the attached helper segment 170b onto the cDNA strand. Subsequent amplification with P7 and P5 primers yields a sequenceable fragment without the need for fragmentation during indexing. This configuration also produces exponential amplification of the signal but only linear amplification of noise, thus significantly improving the signal-to-noise ratio (SNR). Therefore, the incorporation of auxiliary segments 170a and 170b can reduce more than one step to a single step and simplify the cleaning process that must be performed in other cases (e.g., where the desired product will be coupled to the composition after using composition 100).

[0075] However, in other variations, the preparation auxiliary section 170 may additionally or optionally include other sequences configured to reduce steps (e.g., manual steps) associated with the operation (e.g., for a particular platform, for a particular process, etc.).

[0076] 2.2.5 Molecular-Active Region like Figure 1 As shown, each of one or more molecules 120 may optionally include an active segment 180, which functions to enable desired processes (e.g., binding interactions to achieve tagging or synthesis associated with nucleic acid molecules, proteins, etc.).

[0077] In the variant forms, such as Figure 6AAs shown, the active region 180 of one or more molecules 120 may be adapted for mRNA binding, and cDNA synthesis may include one or more of the following: a first sequence 180a for mRNA binding, such as a PolyT sequence (e.g., dTVN or TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN sequence), which enables the capture of mRNA species by interaction with PolyA; and a second sequence 180b for interaction with cDNA synthesized from the captured mRNA (e.g., an rGrGrG group for interaction with the CCC region of the synthesized cDNA added by reverse transcriptase, other groups for interaction with other regions of the synthesized cDNA added by reverse transcriptase, etc.). During the operation, the RT enzyme can terminate cDNA synthesis by adding a CCC sequence (or other sequences), followed by denaturation and removal of the template mRNA. This cDNA sequence can interact with a GGG group (or other complementary group) in the second sequence 180b, where the second sequence is blocked from elongation at the 3' end by a phosphate ester or other suitable blocking group (e.g., C3 spacer, dideoxynucleotide, etc.). Specific sequences other than CCC or GGG can be incorporated into the oligonucleotide tag attached to the bead to provide specific molecular interaction functions and can contain DNA bases, RNA bases, or other groups. Figure 6A As shown, one or more molecules may comprise a first subset and a second subset, the first subset comprising a first sequence (e.g., having sequence 180a) for mRNA binding and the second subset comprising a second sequence (e.g., having sequence 180b) for interaction with cDNA, such that the synthesized cDNA product can be captured and purified on the particles of composition 100 without subsequent purification steps; however, in other variations, the first sequence 180a and the second sequence 180b may alternatively be coupled to different particles. In other variations, the second sequence may not be 3' closed and may extend to the cDNA sequence, forming a complementary sequence to the first strand sequence.

[0078] In such Figure 6AIn other variations shown, the active region 180 of one or more molecules 120 can be adapted to bind a specific target sequence to mRNA, DNA, or other nucleic acid targets, and the synthesis can include one or more of the following: a first sequence 180c for target binding, such as a TotalSeqC capture sequence (e.g., TTTTCTTATATGGG), which enables the capture of oligonucleotide tags attached to antibodies or other target-binding oligonucleotides (e.g., targeting primers) such as targeting one or more mRNA species, gDNA sequences, or specific portions of other sequences; and a second sequence 180b for interaction with DNA (or cDNA) synthesized from the captured nucleic acid. During the operation, the RT enzyme can be terminated after templated cDNA synthesis by adding a CCC sequence (or other sequence), followed by denaturation and removal of the template mRNA. The cDNA sequence can interact with a group containing GGG (or other complementary group) in the second sequence 180b, wherein the second sequence is blocked from elongation at the 3' end by a phosphate ester or other suitable blocking group (e.g., a C3 spacer region, dideoxynucleotide, etc.). Specific sequences other than CCC or GGG can be incorporated into the oligonucleotide tag attached to the bead to provide specific molecular interaction functions, and can contain DNA bases, RNA bases, or other groups. For example... Figure 6A As shown, one or more molecules may comprise a first subset and a second subset, the first subset comprising a first sequence (e.g., having sequence 180c) for targeting nucleic acid binding, and the second subset comprising a second sequence (e.g., having sequence 180b) for nucleic acid hybridization, the second sequence having a universal (e.g., rGrGrG) binding motif or other specific targeting oligonucleotide sequence, such that the resulting synthetic product can be captured (e.g., between known sequence elements of 180c and 180b) and purified on the particles of composition 100 without subsequent purification steps; however, in other variations, the first sequence 180c and the second sequence 180b may alternatively be coupled to different particles. In other variations, the second sequence is not 3' closed and may extend directly to the newly synthesized sequence and form a complementary sequence to the first strand sequence.

[0079] In the implementation plan, there exists such as Figure 6A Two different oligonucleotide tags in the same particle can be configured to provide additional benefits. Figure 6AThe created strand contains oligonucleotides plus cDNA, which then continues as the complementary sequence of the second strand, ultimately capturing CBCs twice, with the second strand being the inverse complementary sequence. In such a case, more than one barcode region originating from the same bead is physically connected, providing a means to improve data analysis (i.e., barcode regions should “match”). However, mismatched barcode regions indicate errors (e.g., if the barcode difference is large, it indicates in vitro recombination; if the difference is only 1-2 bases, it indicates other errors). Therefore, this allows for the identification and potential correction of small errors, and thus the improved ability to map cDNA sequences to the correct beads and, consequently, the correct cells. More specifically, such a configuration provides a second point to provide a degree of error correction. Furthermore, when barcodes do not match sufficiently, these sequences can be excluded from the analysis (or initially assigned to one or another barcode region). Another advantage is that the proportion of this type of chimerism in the data can be measured, and this data can then be used to correct for data that may not be directly measurable. For example, if one uses beads with only one barcode area as beads with multiple barcode areas in the same workflow, one can infer the chirp rate of a barcode area case from the data generated by beads with each sequence assigned to two barcode areas. It is not necessary for both barcodes to be identical for a match to occur. The advantage of "matching" is still possible if the barcodes are constructed differently, but the association is known.

[0080] In other variations, such as Figure 6B As shown, the active region 180 of one or more molecules 120 can be adapted for protein tagging and other processes, and may contain one or more of the following: a third sequence 180c for binding to an antibody (or other protein component) of a target protein, such as an oligonucleotide-antibody binding region (e.g., TotalSeq). TM The RT enzyme contains a CCC region (e.g., a surface antibody) capable of binding antibodies from lysed cells; and a fourth sequence 180d for interacting with the product derived from the capture protein (e.g., an rGrGrG group for interacting with the CCC region added during synthesis, other groups for interacting with other regions added during synthesis, etc.). During operation, the RT enzyme can be terminated by adding a CCC sequence (or other sequence) during synthesis, and the synthesized protein product can then interact with the GGG group (or other complementary group) of the fourth sequence 180d. Figure 6BAs shown, one or more molecules may comprise a first subset and a second subset, the first subset containing a first sequence for antibody binding (e.g., having sequence 180c), and the second subset containing a second sequence for interaction with the synthetic product (e.g., having sequence 180d), such that the synthetic product can be captured and amplified on the particles of composition 100 without subsequent purification steps; however, in other variations, the third sequence 180c and the fourth sequence 180d may alternatively be coupled to different particles. Note that the purification or enhancement of some specific products is achieved by amplifying a specific oligonucleotide sequence over other sequences.

[0081] The composition may additionally or optionally contain one or more other active segments of molecule 120 for carrying out other processes including binding / other interactions.

[0082] 2.2.5.1 Split-type connector For example, such as Figure 6C-6E As shown, one or more cleavable fluorophore quencher regions can be incorporated into the active region 180', which can serve to confirm the cleavage of the oligonucleotide from the host cell based on the emitted fluorescence signal. Figure 6C As shown in the upper right figure, one or more molecules of the composition may include a connector 130' of the coupling molecule to the body 110' (as described above); an active region 180' containing a cleavable element (e.g., a cleavable base or connector) having a fluorophore 180a' and a quencher 180b'; a PCR handle 140'; a barcode region 150'; and a unique molecular identifier (UMI) 160' having a capture sequence.

[0083] During use, such as Figures 6C-6D As shown, biotinylated nucleotides can be incorporated during reverse transcription, where complementary RNA / DNA hybrid strands are generated on some molecules, while others may not capture any target oligonucleotides. A cleavage signal (e.g., a change in ambient temperature to 94°C, or another suitable temperature for the thermally unstable linker) then leads to the cleavage of the thermally unstable linker at the 180' active region, and the release of the complementary RNA / DNA hybrid strand from the molecule containing the RNA / DNA hybrid strand. Figure 6D As shown, after the thermally unstable base / linker separation, quencher 180b is released, allowing fluorophore 180a to emit fluorescence upon excitation. Therefore, the fluorescence signal emitted by fluorophore 180a can confirm the cleavage of the oligonucleotide molecule from host 110.

[0084] More details Figure 6DAs shown, heating results in the presence of multiple molecules in the reaction environment: 1) reverse-transcribed oligonucleotides containing a barcode region 150' and a unique molecular identifier 160' of biotinylated nucleotides; 2) naked / empty / uncaptured oligonucleotide sequences; and 3) RNA-DNA hybrid complementary strands. The separation of the reverse-transcribed oligonucleotides for downstream processing and second-strand synthesis, as well as library preparation, is permitted by removing the liquid phase from the reaction environment, combining the liquid phase with separating particles (e.g., streptavidin magnetic beads, as described in the application incorporated herein by reference) and subsequently separating (e.g., by magnetic force).

[0085] Although a thermal instability mechanism is described, the active region 180' may additionally or optionally include other cleavable mechanisms, thereby allowing product detection to confirm cleavage. For example, the active region 180' may additionally or optionally include a photocleavable region, a chemically cleavable region, an enzymatically cleavable region, or a region cleavable by other suitable mechanisms.

[0086] Furthermore, as described above, the fluorophore 180a' and quencher 180b' can be reverse-oriented in order to monitor lysis and / or capture by means of emitted fluorescence signals.

[0087] In related variations, the active region 180' may alternatively contain a fluorophore, which acts as both a fluorophore and a quencher. Specifically, when the density of fluorophores on the beads is high enough for self-quenching, the removal of some fluorophores from the beads can lead to an increase in total fluorescence, even without the presence of specific quencher molecules. Therefore, even if the number of beads and fluorophores being monitored remains constant, lysis can be monitored by an increase in fluorescence (e.g., fluorescence from the beads or from the pores containing the beads and / or fluorophores released from the supernatant).

[0088] Alternatively, in another variation of the active region 180', the quencher 180b may not be a dark quencher, but rather another fluorophore (e.g., a FRET chaperone) that affects the detection of a signal from the reaction during operation. For example, the active region 180 may incorporate a first fluorophore (e.g., fluorescein) and a second fluorophore (e.g., TAMRA), the first fluorophore being retained on a portion of the body 110 after being configured for cleavage, while the second fluorophore (e.g., TAMRA) is configured to be released via cleavage. This would result in quenching of the fluorescein signal from the first fluorophore when close, but an increase in signal when the oligonucleotide carrying the second fluorophore is released. Furthermore, the signal from the second fluorophore can be monitored in both cleaved and uncleaved configurations.

[0089] exist Figure 6E In one alternative configuration shown, the composition can be configured for direct quantification of beads (e.g., quantification by both full-length and lysed molecules). More specifically, one or more molecules of the composition may include a connector 130” (as described above) that couples the molecule to a body 110”; an active region 180” containing a lysable element with a first fluorophore 180a” (e.g., fluorescein, Cy3, etc.) and a second fluorophore 180b” (e.g., TAMRA, Cy5, Cy7); and additional elements configured as needed for the specific application, such as a PCR handle 140”, a barcode region 150”, and a unique molecular identifier (UMI) 160” with a capture sequence. Such a configuration can be used for direct quantification of the lysed portions of the composition, wherein the composition components can be visualized using different wavelength schemes (e.g., by fluorescence microscopy, by a fluorescence reading device) to alternately detect unly lysed elements (where FRET chaperones remain close together) and lysed elements (where FRET chaperones separate and no longer interact), or preferably only one of the two. The same or similar compositions can be used for quantification without visualization (e.g., for shelf-life testing). Furthermore, such compositions can be used with a body 110 comprising a hydrogel, wherein the hydrogel material used for the body 110 is translucent and does not fluoresce.

[0090] However, other configurations or combinations of the described configurations can be envisioned.

[0091] 2.2.6 Molecular-Molecular Scissors like Figure 1 and Figure 7 As shown, molecular variations of one or more molecules may additionally or optionally include one or more optional molecular scissor regions 190, which function to enable the controlled cleavage of products or other target molecules from one or more molecules 120 (e.g., at some point during biological material treatment, post-synthesis, post-reaction, post-product formation, etc.). Regarding the described embodiments, variations, and examples, molecular scissors broadly include not only specific USER enzyme blends from NEB, but also restriction endonucleases, zinc finger nucleases, TALENs, aptamers, transposases, RNase H, CRISPR enzymes, and other molecules capable of recognizing specific oligonucleotide (e.g., natural or non-natural) sequences and cleaving them at specific locations within the sequence. In variations, the molecular scissors may be single-stranded or double-stranded. In variations, the molecular scissor regions 190 are preferably positioned along the oligonucleotide molecule in regions where cleavage will not damage the desired product or render the desired product unusable (e.g., immediately adjacent to the distal end of the linker). However, the molecular scissor regions 190 may optionally be positioned in other suitable manner. Figure 7(The figure above) depicts an example in which the unit of the composition includes a first molecular scissor region 190a located distally to a first linker 130a along a first oligonucleotide molecule for mRNA capture, and a second molecular scissor region 190b located distally to a second linker 130b along a second oligonucleotide molecule for capturing a synthesized cDNA product. This example allows for the individual, controlled cleavage of mRNA-capturing oligonucleotides from cDNA-targeting oligonucleotides. Figure 7 (The middle image) depicts an example in which the unit of the composition comprises a first molecular scissor region 190a located distally to a first oligonucleotide molecule for mRNA capture adjacent to a first linker 130a, and a second oligonucleotide molecule for capturing synthesized cDNA products. This example allows for controlled cleavage of the mRNA-capturing oligonucleotide. Figure 7 (The figure below) depicts an example in which the unit of the composition includes a first molecular scissor region 190a located distally to a first oligonucleotide molecule for mRNA capture adjacent to a first connector 130a, and another example of a first molecular scissor region 190a located distally to a second oligonucleotide molecule for capturing a synthesized cDNA product adjacent to a second connector 130b. This example allows for the simultaneous cleavage of mRNA-capturing oligonucleotides and cDNA-targeting oligonucleotides.

[0092] Regarding mRNA-cDNA synthesis reactions, molecular scissors can be configured to cleave the product before or after denaturation to remove mRNA. Therefore, the molecular scissor region 190 can be used to remove both the mRNA-cDNA product, the target mRNA, and / or the synthesized cDNA product (without mRNA).

[0093] In one exemplary embodiment, double-strand-specific molecular scissors can be implemented such that the strand is released only after the polymerase extension or reverse transcription or a similar process has completed the second strand. In this way, unreacted products can be washed away, and the completed products can then be selectively released and recovered without generating background contamination from one or more molecules 120 or other portions of composition 100. Figure 7 In alternative variations of the illustrated composition, the bottom molecule providing the aforementioned function may be omitted. In another variation, a molecule having a primer as the active moiety may provide the desired function.

[0094] In addition, in alternative embodiments, one or more molecules 120 and / or other portions of composition 100 may include regions designed for controlled cleavage of oligonucleotide sequences and / or other products using other mechanisms (e.g., photolysis, thermal cleavage, chemical cleavage, etc.).

[0095] 2.2.7 Composition Variation—Functional Molecules Coupled to the Substrate like Figures 8A-8C As shown, variations of the composition can be configured to attach one or more molecules 120 to a substrate 110b for capturing and / or treating target material (e.g., from single cells) (e.g., as a chamber wall, a lid covering a chamber, a protrusion into the chamber, etc.). Variations of the composition and method can also be applied to the methods and compositions described in U.S. Patent No. 10,3891,492, issued August 27, 2019, which is incorporated herein by reference in its entirety.

[0096] More in detail, such as Figure 8A and Figure 8B As shown, the particulate composition can be configured to deliver functionalized oligonucleotide molecules to a substrate 110b (e.g., the wall of a reaction chamber), wherein the molecules coupled to the particulate body contain reactive groups 6 (e.g., at the ends) configured to attach the oligonucleotides to a surface coating 191 of the substrate. In a non-limiting example, the surface coating may contain acrylamide or a similar compound, and the functional linker attached to the oligonucleotides may contain an acrydite modification. Thus, attaching oligonucleotides to the pore surface may include polymerizing more than one acrylamide and acrydite molecule. In some embodiments, the acrylamide polymer may contain a crosslinking agent (e.g., bisacrylamide) or a reversible crosslinking agent (e.g., [bis(acryloyl)cysteine], BAC). In some embodiments, the polymer matrix may be polymerized such that the oligonucleotides are directly attached to the pore wall via covalent bonds. In other embodiments, the attachment may be indirect. For example, in one embodiment, the oligonucleotides may be attached to the wall surface by incorporation into the matrix rather than by direct attachment. In this configuration, the cross-links resulting from the polymerization with BAC are intact, and the oligonucleotides remain functionally attached to the wall. However, as the amount of BAC decreases, the cross-links become unstable, and more than one oligonucleotide subsequently escapes from the surface into the solution. Figure 8A The configuration shown, and in Figure 8B-8M In the subsequent configurations shown, other exemplary surface coating chemistry and / or functional connector chemistry may be implemented.

[0097] like Figure 8B As shown, the reactive group 6 of the chain can pair with the complementary chain 7 via a hybrid oligonucleotide covalently attached to the host 110, and the release of the chain with reactive group 6 from the host 110 prepares it for attachment to the substrate 110b. Regarding the use of the complementary sequence of the oligonucleotide attached to the particle to extract the full-length oligonucleotide from... Figure 8A and Figure 8BThe shown host 110 (e.g., particle 110) is transferred to the pore surface. This complementary sequence can be constructed using primers with a reactive portion at the 5' end (e.g., outside the pore, inside the pore) (e.g., this can be done in batches on more than one host / bead), wherein beads are added to the pore, followed by denaturation to release the complementary oligonucleotides and bind these oligonucleotides to the pore. The application of biotin / streptavidin can provide the desired binding result through multiple rounds of denaturation (e.g., from 1 to 5 rounds), such that the oligonucleotides re-annealed in the first round can detach in subsequent rounds and bind to the available streptavidin on the surface of the substrate 110b.

[0098] like Figure 8C As shown, full-length oligonucleotides for attachment can be delivered into a pore 9 in the form of droplets 8, and the oligonucleotides can be released from the droplets for attachment to a substrate 110b (i.e., the pore surface). The droplets can be liquids in the air (e.g., delivered by a liquid handling subsystem) or bound to various materials (e.g., in an emulsion, such as an aqueous solution containing or without surfactants or other materials bound to an oil). The droplets can be entirely liquid or may optionally contain a hydrogel. For water-in-oil droplets, the oligonucleotides can be released by adding detergents or chemicals that disrupt the emulsion. A non-limiting example is an aqueous droplet bound to an oil that forms a solid (e.g., wax) structure at lower temperatures but reverts to a fluid state at normal biological temperatures.

[0099] Optionally, Figure 8D-8M Variations of attachment processes for surface coupling and / or construction of full-length oligonucleotides on substrate 110b are described.

[0100] In the first variation, such as Figure 8D As shown, common stub oligonucleotides can be provided in solution and attached to substrate 110b (e.g., wall surface), and then constructed from substrate 110b using suitable methods (e.g., using particles, beads, droplets, etc.) to produce full-length oligonucleotides.

[0101] Figure 8E A variation of this construction, which is sequentially built from the surface of substrate 110b, is described, in which an initial residual oligonucleotide is attached to the surface within a pore as described above, and additional oligonucleotide segments or templates (e.g., on particles) are delivered within the pore to extend the attached oligonucleotide to a full-length functional oligonucleotide.

[0102] Figure 8FThe mechanism by which attached oligonucleotides can be extended is described. More specifically, the initial residual oligonucleotide attached to the inner surface of the pore can be extended by the delivery of additional oligonucleotide segments on the particle, wherein these additional oligonucleotide segments are cleaved from the particle (e.g., by chemical means, thermal means, photolysis means, etc.), and the cleaved oligonucleotide segments are subsequently attached to functional linkers of the residual oligonucleotides on the pore surface.

[0103] Figure 8G Depicting Figure 8F A first variation of the mechanism shown involves an additional oligonucleotide segment initially coupled to the particle containing a reactive group configured to attach to a corresponding functional linker upon cleavage from the particle via denaturation. In examples, the reactive group may contain a 5' phosphate for attachment, but may alternatively contain an alkyne or azide for click chemistry, or may alternatively contain other reactive groups (e.g., carbamates, etc.). Figure 8G Depending on the type of reactive group / functional adapter chemistry, the reactive group / functional adapter can be positioned at a 5' or 3' orientation (e.g., configured to react with the 5' phosphate on the functional adapter). Furthermore, the oligonucleotide can be single-stranded or double-stranded, wherein examples of double-stranded oligonucleotides with reactive groups include... Figure 8H As shown.

[0104] Figure 8I Depicting Figure 8G and Figure 8H An alternative variation of the mechanism shown involves the cleavage of oligonucleotides to the cleavable portion of the particle, generating reactive groups that subsequently attach to functional linkers on the pore surface. Similarly, as... Figure 8IAs shown, the 5' and 3' orientations are not specifically referred to. In a non-limiting example, the reactive group could be the 5' phosphate generated after cleavage, wherein the 5' phosphate reacts with a functional linker at the 3' end of the oligonucleotide attached to the pore surface. In one such example, the oligonucleotide on the particle can be constructed with its 5' end attached to the particle and contains a dU residue or a baseless site, which is cleaved by treatment with uracil DNA glycosidase, followed by treatment of the cleaved backbone with a lysin (e.g., endonuclease III, endonuclease VIII) to produce the 5' phosphate. The cleavage product is then prepared for ligation to a usable 3' OH (e.g., the 3' OH at the 3' end of the oligonucleotide attached to the wall surface). In operation, the attachment to the functional linker can be implemented using a splint to facilitate ligation. In variations, the functional linker can be configured as a partially double-stranded construct to serve as a splint, and the oligonucleotide on the particle can be a double-stranded product cut on both strands (e.g., by shifting two dU bases) to produce the desired single-stranded overhang, or additional oligonucleotides can be added separately to serve as splints.

[0105] Figure 8J Depicting Figure 8G-8I An alternative variation of the mechanism shown involves cleavage of the oligonucleotide coupled to the cleavable portion of the particle, releasing the oligonucleotide from the particle for annealing to the 3' end of the functional linker, followed by extension using a polymerase. Figure 8J In the variation shown in the lower right figure, when the geometry, deformability, or density of functional linkers of the particle is good enough that the oligonucleotide can remain attached to the particle without having to release it from the particle.

[0106] Figure 8K Depicting Figure 8G-8J An alternative variation of the mechanism is shown, in which single-stranded oligonucleotides are released from the particle and subsequently annealed to the 3' end of a functional linker on the pore surface for elongation using a polymerase. Figure 8K In the variation shown in the lower right figure, when the geometry, deformability, or density of functional linkers of the particle is good enough that the oligonucleotide can remain attached to the particle without having to release it from the particle.

[0107] Figure 8L Depicting Figure 8G-8K An alternative variation of the mechanism is shown, in which complementary sequences of oligonucleotides are constructed on the particle, and these complementary sequences are used as templates to extend functional linkers on the pore surface. More specifically, the complementary sequences can be constructed by annealing and extending primers at the oligonucleotides on the particle to form the complementary sequences, followed by denaturation to release the complementary sequences from the particle. The functional linkers can then be extended to generate full-length oligonucleotides on the pore surface.

[0108] Figure 8M An exemplary mechanism by which additional oligonucleotide segments can be added to functional linkers coupled to pore surfaces is described. More specifically, this can be achieved by sequentially cleaving oligonucleotide segments from particles and attaching them to a running build of oligonucleotides, thereby adding 1 to... n An additional section is attached to the surface of the hole in this running construct. Regarding Figure 8M The aforementioned attachment methods can be used consecutively, individually, or in combination. For example, each oligonucleotide segment can be added via click chemical linking or attachment, each oligonucleotide segment can be added via extension after hybridization with a template, or some oligonucleotide segments can be added via extension while others can be added via ligation.

[0109] However, Figure 8A-8M The methods and configurations shown may include other steps or elements, some of which will be described in more detail in the following sections.

[0110] 3. Specific examples of the composition—ATAC sequencing, molecular scissors, restriction sites like Figure 9A and Figure 9B As shown, variations of one or more molecules 120 can be configured for transposase accessibility chromatin assays (ATAC-seq) using sequencing to assess genome-associated chromatin accessibility (e.g., for epigenomic analysis). Figure 9A As shown, an example of composition 200 may comprise a body 210, a adapter 230 coupled to the body, a first molecular scissor region 290 coupled to the adapter, a PCR primer 240 coupled to the first molecular scissor region 290, a barcode region 250 coupled to the PCR primer 240, a UMI 260 coupled to the PCR primer 240, and an active segment 280 coupled to the UMI 260, the active segment 280 containing a sequence complementary to a transposase adaptor (e.g., Tn5 transposase 1, Tn5 transposase 2) for ATAC-seq. This configuration is set to perform an initial extension reaction, wherein another transposase adaptor is used for downstream PCR enrichment of an insertion event associated with the first transposase adaptor.

[0111] like Figure 9B As shown, such composition 200 can be configured to cut DNA sequences about chromatin segments, extend them by adding adaptors (and barcodes linked to transposase adaptors) to each end of each fragment, and then amplify and sequence them.

[0112] Figure 9AThe variations of the illustrated example can be configured in other suitable ways. For example, in a second configuration, one or more molecules may include one or more molecules comprising: a connector 230 coupled to the host, a first molecular scissor region 290 coupled to the connector, a PCR primer 240 coupled to the first molecular scissor region 290, a barcode region 250 coupled to the PCR primer 240, a UMI 260 coupled to the PCR primer 240, and an active segment 280 coupled to the UMI 260 containing a first transposase adaptor (e.g., Tn5 transposase 1); and a second one or more molecules comprising: a connector 230 coupled to the host, a first molecular scissor region 290 coupled to the connector, a PCR primer 240 coupled to the first molecular scissor region 290, a barcode region 250 coupled to the PCR primer 240, a UMI 260 coupled to the PCR primer 240, and an active segment 280 coupled to the UMI 260. 260 contains an active segment 280 comprising a second transposase adaptor (e.g., Tn5 transposase 2). This configuration is designed for extension and PCR enrichment on the same particle as composition 200.

[0113] exist Figure 9C In the alternative variations shown, composition 200' can be configured to include cleavable elements 235' and 290', which can be used for the controlled release of oligonucleotides from the host. More specifically, restriction endonucleases can be used to specifically cleave DNA, but require double-stranded segments for cleavage; however, the methods described herein typically utilize single-stranded nucleic acids. Therefore, in order to use restriction endonucleases, it is generally necessary to add a second nucleic acid to the single-stranded molecule to form a double-stranded element for targeted cleavage. This process can be difficult and may result in incomplete cleavage. Composition 200' can be configured to encode at least one cleavage site, wherein one or more molecules may comprise a linker 230' coupled to the host (e.g., a long, flexible linker, such as a spacer region 18 (HEG) sequence providing length and flexibility for bending), a single-stranded sequence encoding a restriction site 290' (e.g., type II restriction endonuclease, type I restriction endonuclease, type IIG restriction endonuclease, type IIP restriction endonuclease, type IIS restriction endonuclease, type III restriction endonuclease; type IV restriction endonuclease), and optionally modified coding region 235' (e.g., internal deoxyuridine modification encoding), forward primer 240a', reverse primer binding site 240b', and optional fluorescent probe target 295' (e.g., a (FAM)-labeled 5' nuclease probe, other probes). Figure 9CIn the illustrated variations, the oligonucleotide molecule is depicted as a linear strand pointing away from the host surface, and the restriction endonuclease requires antiparallel oriented dsDNA. Furthermore, the oligonucleotide molecule can adopt various conformations that allow oligonucleotides close to each other (e.g., the first molecule of composition 200' and the second molecule of composition 200') to at least transiently form an antiparallel double-stranded structure within the region of the restriction endonuclease recognition sequence, thereby forming a complete restriction site, although lacking significant homology except for the palindromic restriction site sequence. Therefore, after lysis using restriction site 290', the oligonucleotides in solution can be detected, sampled, and quantified using various assays (e.g., by qPCR). In a specific example, restriction site 290' contains a BamHI type II restriction endonuclease from *Bacillus amyloliquefaciens*, which has the ability to recognize short sequences (e.g., 6 bp) of nucleic acids and cleave them at the target site. However, other restriction endonucleases can be used as described above.

[0114] During experiments based on examples, untreated beads with single-stranded oligonucleotides on their surface (ostensibly) showed the highest number of molecules released by BamHI cleavage (e.g., about twice the number of molecules produced by treatment with double-stranded products generated by reverse primer hybridization and polymerase extension), while beads with ssDNA and denatured with sodium hydroxide shortly before restriction digestion showed lower cleavage, indicating that cleavage depends on the double-stranded state and requires time for re-annealing.

[0115] In these variations, the molecules form the correct double-stranded motifs through transient hybridization between different oligonucleotide chains (i.e., they do not form hairpins or other secondary structures in a single strand). Furthermore, the absence of sequences capable of completing the restriction site in the remainder of the respective molecular chains indicates the need for intermolecular interactions. Additionally, the use of BamHI restriction sites is not only palindromic but also GC-rich to facilitate cleavage; however, other restriction sites can be used, although cleavage efficiency may vary. More specifically, ssDNA can form a loop structure with only a few bases and can typically exhibit a “randomly coiled” conformation, but the 230' linker length and flexibility play a role in enabling oligonucleotide pair matching for targeted cleavage. Furthermore, in these embodiments, it is not required that the two strands attach before each cleavage. For example, due to the cleavage mechanism of the restriction endonuclease BamHI, both strands of BamHI will be cleaved into the same product, but the absence of a base does not completely inhibit cleavage; therefore, a cleaved oligonucleotide can hybridize with an uncleaved oligonucleotide and induce a second cleavage (e.g., a nick) in the previously uncleaved strand, without the need for the addition of an exogenous complementary strand. Thus, the density of oligonucleotides coupled to the host cell affects the reaction rate, but is not strictly necessary for achieving cleavage.

[0116] Figure 9D Another specific example of a cleavable linker is shown, in which the cleavable linker region 230” can be used for controlled release of oligonucleotides from the host. Figure 9D The composition shown incorporates sequence feature 231” into an oligonucleotide, wherein the sequence feature forms a hairpin structure that at least transiently generates a double-stranded element containing a restriction endonuclease recognition / cleavage site (e.g., a Pac I restriction site). Thus, a temporary double-stranded element is formed for intramolecular target cleavage, thereby enabling the release of the corresponding oligonucleotide chain.

[0117] However, segments of the molecule may additionally or optionally include other suitable segments as described, and / or be coupled to body 210 in other suitable ways. As a non-limiting example, Figure 9C and Figure 9E Restriction site 290' and Figure 9D The pyrolytic connector element can be used to provide a controlled pyrolysis element for other compositions described herein (e.g., as...). Figure 1 The molecular scissor portion 190 of the composition 100 and the molecular scissor portion 290 of the construct 200 are depicted in the figure. Figure 6C (The pyrolytic connector element shown or in which the pyrolytic element is indicated or is beneficial in other compositions).

[0118] In one example, the embodiment of composition 200 can be implemented using method 300 for single-cell ATAC sequencing, wherein, as Figure 10 As shown, method 300 includes: capturing a group of target cells in single-cell form in a capture zone of a microfluidic substrate S310; lysing the group of target cells in the capture zone to remove cytoplasm while retaining the nuclei of the group of target cells S320; co-capturing units of composition 200 with single-cell nuclei S330; causing a transposition reaction between the single-cell nuclei and the composition to generate fragmented DNA S340; performing an extension operation using a first transposase adaptor S350; cleaving a portion of the composition containing the barcode region and UMI from the bulk of the composition via a molecular scissors region S360; adding a second transposase adaptor to the fragmented DNA via the extension operation S370; and performing an amplification reaction on the fragmented and treated DNA S380.

[0119] Variations of Method 300 may also include library cleaning and next-generation sequencing loading steps.

[0120] However, variations of method 300 can be implemented in other suitable ways (e.g., using other capture and processing platforms, etc.).

[0121] 4. Preparation like Figure 11As shown, a method 400 for generating the composition includes: providing a host as a base substrate S410; coupling a set of linkers to the host S420; and coupling one or more molecules to the set of linkers S430 via a staged / sequential attachment operation. In embodiments, long (length > 50 bp) oligonucleotide molecules can be prepared using various molecular biology reactions (e.g., ligation or polymerase extension) or chemical synthesis methods (e.g., click chemistry) to obtain very well-defined sequences with minimal error rates (e.g., less than 5% error, less than 1% error, less than 0.5% error). In some instances, these may include templated reactions in which the template for defining the sequence is not directly incorporated into the final product. In other instances, the reaction may be untemplated or carried out in a manner where the template is indeed incorporated. The oligonucleotide may be constructed from component monomer units or by adding partial or complete sequences. In some instances, the added units may be partially single-stranded or fully single-stranded. In other embodiments, the added units are partially double-stranded or fully double-stranded. In some embodiments, the added units are primarily double-stranded, but only one of the two strands is covalently attached to the host and / or adapter. In some embodiments, the added template strand and / or unit undergoes purification or quality control checks before use for attachment, thereby reducing the error rate of the final product by minimizing errors present in the individual units. In some cases, the method of preparing the individual units inherently ensures a reduced error rate (e.g., by using short oligonucleotide units). Figure 11 In some implementations and variations, the steps from the second step to the final step (e.g., coupling a set of connectors to the body S420) can be optional. For example, in step S430, it is possible to potentially attach connectors to each molecule in the set.

[0122] Based on one or more benefits described in Section 1 above, Method 400 is used to efficiently create compositions that allow for the separation and recovery of target materials from sample processing. Method 400 can produce compositions with complex oligonucleotide structures in a staged attachment manner, which reduces composite errors associated with base-by-base oligonucleotide attachment methods (e.g., phosphoramide-based oligonucleotide synthesis). Method 400 can also be used by incorporating molecular adaptors specifically designed for sequencing platforms (e.g., Illumina). TM (e.g., linkers) to produce compositions that simplify the library preparation process. Therefore, method 400 can be used to prepare functionalized particles in a scalable manner, and to prepare functionalized particles in a way that provides quality control and increases the amount of recyclable product.

[0123] In the embodiments, method 400 can produce the embodiments, variations, and examples of compositions 100 and 200 described above. However, portions of method 300 can be applied to produce other related compositions.

[0124] Box S410 describes providing a host as a basic substrate, the function of which is to provide a basic substrate for attaching functional molecules specifically for various processes. As described above, the substrate can be provided as a continuous host, or optionally as a cluster of smaller hosts. In either continuous or clustered form, box S410 may include coupling of functional groups (e.g., amines, hydroxyl groups, silanol groups, etc.) to the host to facilitate subsequent attachment of linker molecules to the host surface.

[0125] In an alternative variation, as described above, box S410 may include aggregating a group of smaller entities to form a main body. In the first variation, such as Figure 12B As shown, block S410 may include S414, which uses a microfluidic channel to generate droplets of unpolymerized and / or uncrosslinked material, whereby the material polymerizes and / or crosslinks in droplet form to form a set of smaller bulks. According to block S414, the material can flow through the microfluidic channel at a desired rate and enter a medium (e.g., oil) through openings of the desired morphology to generate droplets of the desired size. Polymerization can then be achieved by chemical or other means. Similarly, crosslinking can be achieved using one or more of the following methods: photoactivated methods, chemical methods, thermally induced methods, and / or any other suitable method.

[0126] In another alternative form of variation, such as Figure 12C As shown, block S410 may include distributing a set of smaller bodies throughout a substrate in a prepolymerized aqueous solution in a pore structure ... Frame S416 may additionally or optionally include crosslinking (e.g., crosslinking by irradiation, chemical crosslinking, thermal crosslinking, oxidative crosslinking, etc.).

[0127] However, other variations of box S410 may include additional or alternative steps for forming a set of smaller host clusters with appropriate surface chemistry (and / or core material characteristics, such as magnetism) to provide a substrate for oligonucleotide functionalization.

[0128] In a first variation, box S410 may include generating a base substrate in the form of beads, wherein the beads consist of a polymer dissolved in a controlled environment. In a particular example, the beads may consist of a polyacrylamide material obtained by treating an acrylamide solution (e.g., 40% v / v acrylamide, other percentages of acrylamide), bis(acryloyl)cysteine ​​(e.g., 0.8% w / v BAC, other percentages of BAC), deionized water, and a buffer (e.g., a buffer containing Tris-HCl, NaCl, KCl, EDTA, Triton X-100 and water, other suitable buffers, etc.), wherein the polyacrylamide beads are configured to polymerize with ammonium persulfate (e.g., 10% APS, other percentages of APS) and tetramethylethylenediamine (TEMED) under hypoxic conditions (e.g., under argon) and subsequently dissolved in the presence of a reducing agent such as dithiothreitol (DTT).

[0129] In this variation, such as Figure 12A As shown, the bead generation according to frame S410 includes: transporting material components and an initiator into a first microfluidic path S411; providing TEMED to the oil phase during collection while pumping (e.g., with a pressurized gas pump) the material obtained in S411 through a second microfluidic path (e.g., terminating in a 14µm focusing channel with a 500µl collection volume), generating a set of droplets with the obtained material S412; and controlling the droplet size of the set of droplets based on the characteristics of the microfluidic channel and the gas composition (e.g., argon, other gases) used to pump the material components through the microfluidic path S413. In a specific example of S410-S413, a pressurized pump with pressure and flow control (e.g., pressurized argon to pressurize and remove air from the pump chamber for hydrogel polymerization) is coupled to a first microfluidic chip containing the first fluid path and a second microfluidic chip containing the second fluid path, wherein the quality and size of the formed droplets are monitored using an XY stage and a high-speed camera mounted on a microscope controlled by a flow control center. In this example, the formed polyacrylamide droplets were washed with a buffer solution containing Tris-HCl, NaCl, KCl, EDTA, Triton X-100, and water, and then placed in a Tris Tween-20 storage solution, wherein the formed droplets had an average diameter of 22.75 μm (e.g., swelling in aqueous solution) and a standard deviation of 1.62 μm. In this example, the droplets dissolved within 30 seconds in 0.1 M DTT at a 1:1 volume ratio.

[0130] In Figure 12A In a variation of a related example, the polyacrylamide bead formulation was adjusted by reducing the amount of acrylamide and adding an acrylamide-tagged (e.g., acrydite-modified) oligonucleotide to provide approximately 10 [units of something] per bead. 9 Oligonucleotides. In some variations, oligonucleotides are further modified (e.g., with fluorophores or other modifications) for fluorescent tagging and detection applications. More specifically, the beads may comprise a polyacrylamide material obtained by treatment with an acrylamide solution (e.g., 40% v / v acrylamide, other percentages of acrylamide), bis(acryloyl)cysteine ​​(e.g., 0.8% w / v BAC, other percentages of BAC, deionized water), acrydite-treated oligonucleotides (e.g., 250 μM acrydite-treated fluorescein imide (FAM) oligonucleotides having an acrydite-treated site at the first proximal end and a FAM-treated site at the second proximal end), ammonium persulfate solution (e.g., 10% w / v APS, other percentages of APS), and buffers (e.g., buffers containing Tris-HCl, NaCl, KCl, EDTA, Triton X-100, and water, other suitable buffers, etc.), wherein the FAM-tagged polyacrylamide beads are configured to polymerize with tetramethylethylenediamine (TEMED) and dissolve in a dithiothreitol (DTT) solution. In an example of fluorescently tagged beads, the formed droplets have a diameter of 20.39 μm. The droplet has an average diameter of 1.25 μm (e.g., swollen in an aqueous solution) and a standard deviation of 1.25 μm. In the example, the droplet is soluble in 0.1 M DTT at a 1:1 volume ratio (e.g., by imaging at 0 sec, 30 sec, 90 sec, and 5 min), where the fluorescence signal indicates the dissolution process. In this non-limiting example, DTT breaks the disulfide crosslinks present due to the BAC element, thereby releasing a smaller bulk (e.g., a polyacrylamide-linked oligonucleotide) from the spherical bead. The smaller bulk has a size that allows it to diffuse easily through the solution. However, variations of this non-limiting example can also be implemented.

[0131] Box S420 describes the coupling of a set of adapters to a host, wherein the adapters function to control the spacing and density of the set of oligonucleotide molecules coupled to the host to achieve functionalization of the composition. In embodiments, the adapters may be embodiments, variations, or examples of the adapter 130 described above; however, the adapters may be other suitable adapters.

[0132] In variations including asymmetric linkers (e.g., linkers with branches of different lengths or linkers of similar lengths but with different functional groups or protecting groups), box S420 may include constructing a first oligonucleotide segment from a first branch of the asymmetric linker while protecting the second branch with a second protecting group, and constructing a second oligonucleotide segment separately from the second branch of the asymmetric linker while protecting the first branch with a first protecting group, S425. However, variations of box S425 may be configured to operate without a linker or by coupling a synthesized oligonucleotide to the attachment site of the composition.

[0133] Box S430 describes how coupling one or more molecules to this set of linkers via staged / sequential attachment operations reduces recombination errors and batch-to-batch variability typical of the chemical synthesis of oligonucleotide chains. More specifically, Box S430 functions to provide a method that involves fewer addition events to produce fewer recombination errors, resulting in more accurate oligonucleotide molecules relative to the amount of available full-length product (e.g., over 97% of available product), greater control over molecular design, and higher synthetic efficiency. In some embodiments, it is also used to confine incomplete products to discrete units larger than a single substrate, providing the advantage of preventing partial product involvement in downstream workflows and facilitating data analysis that can distinguish preparation errors from artifacts in downstream processes, thus improving subsequent data analysis.

[0134] like Figure 13 As shown, in a variation, box S430 may include a set of sub-segments (e.g., parallel, tandem) S431 that generate the desired oligonucleotide molecule configured for use in the above reaction. Box S430 may then include S432 that assembles the set of sub-segments into the desired oligonucleotide molecule as a full-length product with reduced errors. In some variations, box S430 may include a purification unit S433 for the set of sub-segments to further reduce errors in assembly, wherein purification may include a complete purification process and / or a desalting step. Alternatively or additionally, some variations may include purification-related steps after assembling the desired oligonucleotide molecule; however, some variations of method S430 may omit the purification step associated with box S433. In a variation, the stage attachment method of box S430 includes generating sub-segments of 5-30 bases in length and then assembling them; however, in alternative variations, the stage attachment method of box S430 may include generating sub-segments of other suitable lengths.

[0135] Regarding the barcode segment or other segments described above, such as Figure 14As shown, a specific instance of box S430 may include generating a barcode segment (e.g., a segment approximately 20 bases in length), wherein, as Figure 14 As shown, the barcode segments are selected from a group of 96-384 possible barcode sequence formats. However, other suitable numbers of barcode sequence formats can be generated using sequences of appropriate length that do not naturally exist.

[0136] In specific instances, three types of segments can be generated from barcode sequences with unique overhangs (e.g., with associated identifiers), where the overhangs can be used to assist oligonucleotide molecules in assembling correctly in the desired order. For example, as... Figure 15 As shown, the first barcode sequence 435 may include a protrusion for coupling with the second barcode sequence 436, the second barcode sequence 436 having a protrusion for coupling with the third barcode sequence, and the unique molecular identifier 437 having a protrusion for coupling with the active group 438 (e.g., oligonucleotides TVN, TS GGG, TotalSeq C, etc.). The assembled barcode segment may be coupled to a precursor molecule (e.g., a linker coupled to a primer), which is coupled to the body provided in box S410, or otherwise coupled to a precursor molecule.

[0137] In more detail with respect to specific examples, the precursor of the composition can be constructed using a body (e.g., a bead) coupled to a linker (e.g., a C18 linker) that is coupled to an oligonucleotide containing a primer-binding site (e.g., a TSO primer) followed by a set of bases (e.g., 8 thymine bases). A first barcode segment with protruding ends on each side of the first barcode segment can then be pre-hybridized and then coupled to the precursor of the composition via a suitable ligase. Subsequent barcode segments with protruding ends can be coupled to the running construct of the barcode region until the desired barcode region length is reached. For each step of the barcode segment assembly, a complementary segment containing the detection portion (e.g., a fluorophore segment) can be added as a tag to the current segment being added, wherein detection of the detection portion (e.g., by an optical detection method) can be used for quality control at each step of the stage attachment method. However, quality control at each stage of the stage attachment method can be performed in other suitable ways or omitted.

[0138] Other alternative variations of box S430 may include synthetic operations involving the addition of single bases configured as nucleotides to form an oligonucleotide product. In specific instances of these alternative variations, chemical synthesis involves adding nucleotide bases one at a time to a linker (e.g., a C18 linker) to produce a full-length product. Furthermore, variations of method 400 may include hybridization methods, whereby a portion of the oligonucleotide molecule (e.g., linker and primer segments) is formed through base-by-base synthesis, and the remaining portion of the oligonucleotide molecule is formed through a staged attachment method comprising assembling shorter sub-segments of the oligonucleotide.

[0139] Method 400 may additionally or optionally include other suitable steps. For example, variations of method 400 may include steps related to preparation, scale-up, and quality control to improve the efficiency of generating a usable product, including one or more of the following: reacting with a ligase (e.g., NEB-M0202M) in a controlled environment (e.g., at a concentration required for the number of particles generated) to couple the generated oligonucleotide segments; providing the required concentration of oligonucleotide material according to the number of particles generated; providing the required reaction volume (e.g., in a container with sufficient headroom to allow for washing steps); providing a stabilizing agent (e.g., polyethylene glycol) during preparation to improve reaction efficiency; performing an oscillation procedure (or other procedure to thoroughly disperse or produce a homogeneous product under the desired reaction conditions); performing an incubation procedure (e.g., 16 ± 5 °C or 16 ± 1 °C) during the preparation of the composition; and performing an appropriate number of washing steps. Additionally, variations of method 400 may exclude certain elements from the preparation process, such as preparation with a DTT-free ligase and removal of DTT from other reagents in the process, or other potential release agents (temperature, chemicals, etc.) of smaller components from the preparation process. However, method 400 may additionally or optionally include other suitable steps of the method for mass production of units of compositions 100, 200.

[0140] 4.1.1 First Preparation Example—Next-Generation Barcode Beads In one instance, method 400' can be adapted to create more than one barcode group on each subject using a limited (e.g., a small number) of barcode groups combined in known and unique combinations, where each bead has different combinations of barcode sequences. All combinations of barcode sequences on a single bead can be unique to that bead or can be otherwise configured. Thus, method 400' can implement a limited number of barcode groups combined in known combinations such that a single preparation construct produces more than one barcode (CBC) per bead in a controlled and predictable manner, allowing all these different barcodes to be mapped back to the same bead.

[0141] More specifically, each barcode unit may comprise a subsequence of barcode units having a set of bases (e.g., less than 10 bases to more than 10 bases) and one or more handles (e.g., one of a set of different connecting handles, or one of a set of connecting handles at either end, or other handles, such as polymerase extension handles), wherein the subsequence of barcode units may be configured as a group primarily defined by handles. In variations, each handle may each have between 3 and 15 bases, or other suitable number of bases. Thus, each subsequence of barcode units in an assembly group is configured with the same handle (e.g., one of a set of different connecting handles), while different groups have other handles from that set of different handles. Therefore, the number of connecting handles can be determined based on the number of barcode sequences required for each bead and the desired total barcode diversity.

[0142] In an example, method 400' can implement multiple barcode unit subsequences (e.g., 96 barcode units, 384 barcode units, or other numbers of barcode units), and a set of connecting handles (e.g., 4 connecting handles, fewer than 4 connecting handles, more than 4 connecting handles) to achieve the desired level of diversity for the sample being processed and the different number of barcodes required per bead. Each group can have a unique barcode, but optionally, the same group (e.g., 96 barcode subsequences, 384 barcode subsequences, etc.) can be used for all groups. In one example, 96 barcode unit subsequences with 7-mer barcodes can be implemented using 4-base connecting handles, wherein the barcode unit subsequences are selected from four different groups of the 96 barcode unit subsequences; however, other numbers of barcode unit subsequence groups can be used, including single groups that are distinguished only by the handle sequence.

[0143] Extending this example, to provide four different unique barcode sequences on a single bead, method 400' can implement a first group of barcode sub-sequences with connecting handles ATCG, wherein these sub-sequences are 7-mer barcode sequences (e.g., one of a group of 96 barcode sequences, one of a group of 384 barcode sequences, or one of other numbers of barcode sequences); a second group of barcode sub-sequences with connecting handles TCGA, wherein these sub-sequences are 7-mer barcode sequences; a third group of barcode sub-sequences with connecting handles CGAT, wherein these sub-sequences are 7-mer barcode sequences; and a fourth group 404' of barcode sub-sequences with connecting handles GATC, wherein these sub-sequences are 7-mer barcode sequences. Thus, the connecting handles ATCG, TCGA, CGAT, and GATC are specific to this group, but the sub-sequences may not be specific to this group. In this example, for the first (e.g., ATCG, TCGA, CGAT, and GATC), second (e.g., TCAG, AATC, ATTA, and TCCT), third, and fourth linkage reactions associated with a single bead, the specific four-base linkage handles are different and also different for each set of barcode unit subsequences. Therefore, this configuration provides 16 different handles across four sets of barcode unit subsequences via four linkage events (e.g., the number of handles is the product of the number of barcode unit subsequence sets and the desired number of linkage events).

[0144] In the implementation of method 400', all the first set of barcode variations can be provided in the first hole, all the second set of barcode variations can be provided in the second hole, and so on, in order to generate unique barcoded beads with different barcodes coupled to each bead (e.g., hole 1 contains barcode 1 ATCG, barcode 1 TCGA, barcode 1 CGAT and barcode 1 GATC; hole 2 has barcode 2 ATCG, barcode 2 TCGA, barcode 2 CGAT and barcode 2 GATC, etc.). In optional variations, different barcode variations from each group can be provided in each hole, as long as each hole has a unique identifiable barcode from each barcode group (e.g., hole 1 has barcode 1 ATCG, barcode 25 TCGA, barcode 49 CGAT, and barcode 76 GATC; hole 2 has barcode 2 ATCG, barcode 33 TCGA, barcode 82 CGAT, and barcode 25 GATC; or alternatively, if each barcode group originates from different groups of 96, for example, hole 1 has barcode 1 ATCG, barcode 97 TCGA, barcode 193 CGAT, and barcode 290 GATC, etc.).

[0145] Figures 16A-16DThe sequence of creating beads (i.e., body 110') with four different barcodes is depicted, where each individual bead eventually has a set of four uniquely identifiable barcodes (CBCs) after a set of connection events. Figure 16A As shown, exemplary method 400' may include: adding a first set of barcode unit subsequences S410' with different linker handles to the 3' end, wherein different barcode unit subsequences 411', 412', 413', and 414' in the first set of barcode unit subsequences hybridize with a splice oligonucleotide 415' having the same overlapping sequence. Regarding step S410', each of the first set of barcode unit subsequences can be added together to achieve a desired ratio between the different units (e.g., 1:1:1:1, a non-1:1:1:1 ratio, etc.). The product obtained after the first round of ligation will be four different oligonucleotide chains (or other suitable numbers in other variations) on each bead, each oligonucleotide chain having a different linker handle. In one variation, the barcode unit subsequences within a well can be identical, having different linker handles for distinguishing groups. In another variation, the barcode unit subsequences can be different, but their association is known because they come from the same well.

[0146] like Figure 16B As shown, exemplary method 400' may include: adding a second set of barcode unit subsequences to the corresponding end S420' of the first set of barcode unit subsequences, wherein the second barcode unit subsequence is in Figure 16B The numbers are 421', 422', 423', and 424'. Regarding step S420', each of the second group of barcode unit subsequences can be added together to achieve the desired ratio between different units (e.g., 1:1:1:1, non-1:1:1:1 ratio, etc.).

[0147] like Figure 16C As shown, exemplary method 400' may include: adding a third group of barcode unit subsequences to the corresponding end S430' of the second group of barcode unit subsequences, wherein the third group of barcode unit subsequences in Figure 16C The numbers are shown as 431', 432', 433', and 434'. Regarding step S430', each of the third group of barcode unit sub-sequences can be added together to achieve the desired ratio between different units (e.g., 1:1:1:1, non-1:1:1:1 ratio, etc.). Furthermore, as... Figure 15 As shown in C, the third set of barcode unit subsequences may optionally contain a unique molecular identifier sequence, as described above.

[0148] like Figure 16DAs shown, exemplary method 400' may include: adding a set of capture oligonucleotides to the corresponding ends S440' of a third set of barcode unit sub-sequences, wherein similar capture oligonucleotides are present in... Figure 16D The subsequence is denoted as 441', and different splice oligonucleotides are implemented (i.e., 445', 446', 447', 448'). Although three sets of barcode unit subsequences are described, method 400' may include adding any other suitable number of barcode unit subsequences to achieve the desired diversity. Regarding the exemplary method 400, the result after 3 (or however many) rounds of joining (pooling and separating between rounds) is that the beads have the same barcode diversity we would have for a single barcode sequence, but with 4 different barcode sequences on each bead. The 4 different capture sequences can be placed on these beads using different ends, and since the association of barcode unit subsequences is known, any group of barcodes should not only match at a single barcode position, but should also form aggregated barcode sequences across groups of 3 barcode unit subsequences.

[0149] More specifically, if the same capture sequence is applied to all oligonucleotide chains of a particular bead, even if they contain different composite barcodes, the sequence pooling from any cell will map to one of a limited whitelist of barcode subsequence groups associated with that particular bead, allowing for better identification of sequencing errors or chimeric sequences. The linker used also corresponds to a specific group of all positions of the aggregated barcode sequences from the individual barcode unit subsequences. Therefore, any crossovers of groups can be detected and these sequences can be labeled. In clinical applications, the ability to identify sequences (e.g., transcripts) that are relatively rare (e.g., greater than 1) of captures as originating from the same cell due to the existence of more than one different barcode that all map to the same bead (and therefore the same cell) will greatly increase the certainty of any barcode-related determination and thus the certainty of any potential diagnosis. A specific transcript or group of transcripts associated with a UMI that is different from the aggregated barcode sequence may be different transcripts from a single target cell, but may also be caused by chimeric sequences. Therefore, mapping to four different aggregated barcodes (all of which are associated with a single bead) provides greater confidence that they originate from a single cell.

[0150] Another benefit of using individual groups of barcode unit subsequences according to exemplary method 400' is that the "invariant" connection handle will now be co-associated with each individual bead, exhibiting diversity, and thus avoiding sequencing markers, thereby allowing for more cost-effective use of downstream processes.

[0151] Although three sets of barcode unit subsequences are described, method 400' may include adding any other suitable number of barcode unit subsequences. Regarding the exemplary method 400, the result after 3 (or however many) rounds of connection (converging and separating between rounds) is that the beads have the same barcode diversity we would have for a single barcode sequence, but with 4 different barcode sequences on each bead. The 4 different capture sequences can be placed on these beads using different ends, and because the correlation of the barcode unit subsequences is known, any group of barcodes should not only match at a single barcode location, but should also form an aggregated barcode sequence across groups of the 3 barcode unit subsequences.

[0152] In a variation of method 400', such as Figure 16E As shown, the method may include: adding a set of capture oligonucleotides to the corresponding ends S440' of a third set of barcode unit subsequences, wherein the capture oligonucleotides correspond to Figure 16E The figures 441', 442', 443', and 444' are shown. Step S440" differs from step S440' in that, after the final ligation step, the resulting composition contains more than one different aggregation barcode sequence (CBC) per bead, having the same PCR handle but with a different capture sequence on each aggregation barcode sequence. Therefore, this configuration allows for the simultaneous capture of different targets, with the ability to reliably map back to each cell even if the aggregation barcode sequences are not identical.

[0153] In yet another variation of method 400', such as Figure 17A As shown, the method may include: adding a first set of barcode unit sub-sequences with different linker ends to the 3' end and adding them to different PCR linkers S410", wherein the different barcode unit sub-sequences 411", 412", 413", and 414" in the first set of barcode unit sub-sequences hybridize with a splice oligonucleotide 415" having complementary overlapping sequences. Regarding step S410", each of the first set of barcode unit sub-sequences may be added together to achieve the desired ratio between the different units (e.g., 1:1:1:1, a non-1:1:1:1 ratio, etc.). Then, the mixture is hybridized in a manner similar to that described with respect to steps S420' to S440' above. Figure 17BAs shown, this method can produce bead compositions in which each bead has a different barcode sequence that can be addressed independently (because different PCR handles are applied in step S410). In particular, depending on the application, the final captured oligonucleotides can be the same or different. Furthermore, each final captured oligonucleotide can be addressed individually using a different PCR handle, but can still be mapped back to the same bead. Therefore, even if the sample is processed using different downstream workflows (e.g., after initial capture and extension by reverse transcription or polymerase extension), a connection and association with a specific cell / bead can exist.

[0154] As described above, methods 400' and 400" are shown as attaching an oligonucleotide sequence to a bead; however, methods 400' and 400" can additionally or optionally be adapted to incorporate cleavage sites (e.g., molecular scissor regions, restriction sites, etc.) as described in the various variations above. Furthermore, in some applications, the oligonucleotide can be attached to the bead via its 5' end and has a free 3'-OH group. In other applications, the oligonucleotide can be attached to the bead via its 3' end. In other applications, different barcode sets can contain oligonucleotides assembled to potentially have the same sequence after ligation, but configured such that one barcode set is added by extending the oligonucleotide along the 5' to 3' direction, while the other oligonucleotide is extended along the 3' to 5' direction.

[0155] Regarding the 400' and 400" steps of the above method, keeping the beads suspended during ligation is beneficial for overall ligation and may also improve the uniformity of ligation between beads. The precise speed will vary depending on the size and shape of the container and the number of beads in the reaction. In the example, the relevant mixture was shaken at 1500 RPM in a shaking device; however, other shaking parameters can be implemented. Regarding the time for each ligation step, a ligation time of less than 1 hour may reduce the overall ligation efficiency or require additional enzyme to achieve the same efficiency. In the example, ligation time periods between 4 hours and 24 hours were implemented for each ligation, incubated at 16°C; however, other ligation times and incubation temperatures can be implemented.

[0156] The inherent characteristic of separate and aggregate synthesis methods used for bead preparation is that beads with incomplete oligonucleotides will be grouped together. Therefore, an unlinked barcode from one well may link to an oligonucleotide that would otherwise be on beads in different wells. This is especially true when the number of "stubs" (i.e., incomplete oligonucleotides attached to the beads) is not fully saturated by the barcodes. The result will be beads with more than one type of barcode on the same bead, leading to misallocation of sequence data during analysis. This type of contamination is highly undesirable. Collecting beads (and ligation reaction components) from more than one well into a larger tube, collecting the beads, followed by precipitation to retain the beads and removal of the supernatant, and then washing the beads significantly reduces cross-contamination and mitigates the aforementioned effects (e.g., if operating rapidly). Alternatively, for automated systems or when any beads remain in the intermediate mixed solution, ligation should be inhibited (e.g., by stopping the solution, by heat inactivating the enzyme, by dephosphorylating the barcode oligonucleotide, by adding blocking oligonucleotides, by depleting ATP from the ligation solution, or other suitable methods). An exemplary stop solution may contain EDTA bound with approximately 2 molar equivalents of Mg++.

[0157] The ideal number of oligonucleotides per bead also varies depending on the bead composition and end use. For example, using a submaximal amount of barcode oligonucleotides for ligation can achieve improved performance and reduced cost. An exemplary method implements 850 nanomoles of partially double-stranded oligonucleotides with approximately 3.5 million beads, or approximately 0.25 picomoles per bead, in the ligation reaction. By reducing the amount of partially double-stranded oligonucleotides to 172 nanomoles per 3.5 million beads, or approximately 50 femtomoles per bead, preparation costs are significantly reduced and performance is improved. This example achieves a more optimized distribution of oligonucleotides around each bead, resulting in less steric hindrance, as steric hindrance would cause adjacent oligonucleotides to ligate at a lower rate, resulting in a more dispersed set of full-length oligonucleotides. The amount of ligase is also proportional to the number of beads and the number of ligation events per bead. In this example, 33,333 sticky end units are implemented per 3.5 million beads, or approximately 0.0095 sticky end units per bead.

[0158] Other linker components that can improve the linkage include PEG 6000 to a final concentration of 10% w / v, and Mg++ to a concentration of 10 mM (or by replacing up to ~50% of the magnesium with other divalent cations or a larger amount of monovalent cations, where monovalent = 120 * the square root of [divalent]). Other linker components can be implemented additionally or optionally to create a suitable reaction environment.

[0159] In addition, although the connection is described in exemplary methods 400', 400'', other assembly or extension methods (such as templated polymerase extension or chemical attachment, such as click attachment, etc.) may also be implemented.

[0160] Regarding the list of barcode unit subsequences related to the methods described above, various example lists may contain between 96 (or fewer) and 932 (or more) barcode unit subsequences. In particular, groups can be configured with larger Hamming distances, Levenshtein distances, or other distances to provide properties that are easily corrected by post-sequencing analysis. Groups can also be configured, alternatively, to produce beads with lower overall barcode diversity.

[0161] However, other suitable configurations and / or the number of barcode units per list can be implemented.

[0162] 4.1.2 Second Preparation Example In this example, the preparation method can begin with more than one well (e.g., 96 wells), each containing more than one million microspheres and a unique oligonucleotide segment attached (e.g., by linking) to each bead, under optimal conditions of time, temperature, and oscillation, as well as composition (e.g., enzyme concentration, oligonucleotide concentration, reaction enhancer, providing crowding). After attaching the unique oligonucleotide tag to all particles present in each tube (e.g., 96 tubes), the beads can be washed such that, after washing, no product residue occurs when all the beads from the 96 tubes are pooled together (e.g., 1 million beads per tube x 96 tubes = 96 million beads pooled together).

[0163] After a period of washing, the beads are redistributed into 96 different tubes containing uniquely barcoded oligonucleotide segments, and then additional reagents (e.g., ligases, ATP, PEG, reaction enhancers) are added to continue the second stage of attachment. This process of barcoded segment reaction, washing, collection, and redistribution continues until all the different oligonucleotide segments have been added to complete the process. The liquid handling process for bead separation-collection-washing and reaction can be automated in 96-well plates or in other plate sizes (such as 384-well or 1536-well plates). Reagent dispensing in each well can be done using a liquid pipette or other methods, such as inkjet nozzles or acoustic jetting from an inverted plate. Bead collection can be done using a pipette or by using a specially designed receiving cap that can be placed on a 96-well plate and then inverted and agitated to collect all beads in the receiving cap. The liquid handling operation is designed to minimize contamination throughout the process to prevent any errors from spreading. The invention described herein will allow for a significantly simplified workflow in preparing these barcode beads. The total number of beads that can be prepared can range from as low as 10 million to as high as 10 billion, exhibiting bead diversity with more than 100,000 (or 1 million or >10 million) different unique combinations.

[0164] In some implementations, the unique oligonucleotide present in each well may comprise fragments of different sizes in different wells. For clarity, a specific example could be that 32 wells each contain a partially double-stranded construct comprising 6 bases providing overlap with the preceding segment to facilitate ligation, 7 unique bases defining the barcode segment, and 4 bases providing overlap with the next segment. Another 32 wells contain partially double-stranded constructs comprising the same 6 bases providing overlap with the preceding segment, 8 unique bases defining the barcode segment, and 4 bases providing overlap with the next segment. A third set of 32 wells each contains partially double-stranded constructs comprising the same 6 bases providing overlap with the preceding segment, 9 unique bases defining the barcode segment, and 4 bases providing overlap with the next segment. When used in the above preparation method, this will result in full-length oligonucleotides of varying lengths due to the inclusion of fragments of different lengths. When the sequence of the readable barcode region is subsequently generated, barcodes prepared in this manner will have several distinct advantages in sequence generation and analysis that are not present in typical preparation methods. In particular, when generating more than one sequence from more than one bead, the sequences generated by the above method can possess the beneficial property that some or all of the overlapping portions of the sequences will be identical. Therefore, they can be used as alignment markers and provide additional benefits for analysis, such as identifying chimeric molecules, sequencing errors, or preparation mistakes, among other advantages.

[0165] Typically, if a large portion of the sequence contains all the same bases at a particular position, the sequencer used for these analyses will generate errors and terminate the run, thus failing to collect the desired experimental data. Therefore, when all sequences are of the same length, containing identical sequences, such as the same overlapping regions described herein, can be problematic. By varying the length of the barcode unit preceding the constant region as described herein, the resulting sequence becomes offset. Although overlapping regions or more than one overlapping region can remain substantially unchanged in more than one sequence, they are effectively out of phase, thus allowing the benefit of identical or nearly identical markers to be achieved without introducing errors in the sequencing process itself. This can be achieved in the preparation process described herein with variations in the number of wells or tubes and different configurations of sequence lengths, which are used here for illustration, provided they are suitable for providing the dual benefits of addressing the limitations of sequencing instruments and providing improved post-sequencing analysis.

[0166] 5. Conclusion The accompanying drawings illustrate the architecture, functionality, and operation of possible implementations of systems, methods, and computer program products according to preferred embodiments, exemplary configurations, and variations thereof. In this regard, each box in a flowchart or block diagram may represent a module, segment, or portion of code containing one or more executable instructions for implementing one or more specified logical functions. It should also be noted that in some alternative implementations, the functions mentioned in the boxes may not appear in the order shown in the drawings. For example, two boxes shown consecutively may actually be performed substantially simultaneously, or the boxes may sometimes be performed in reverse order, depending on the functions involved. It should also be noted that each box in the block diagram and / or flowchart illustration, and combinations of boxes in the block diagram and / or flowchart illustration, may be implemented by a dedicated hardware-based system or a combination of dedicated hardware and computer instructions that performs the specified functions or actions.

[0167] As will be appreciated by those skilled in the art from the foregoing detailed description and from the accompanying drawings and claims, modifications and variations may be made to the preferred embodiments of the invention without departing from the scope of the invention as defined in the appended claims.

Claims

1. A composition comprising: -The main body; and - A group of oligonucleotides coupled to the host, wherein one or more of the group of nucleotides comprises: --The joint area coupled to the main body --Single-stranded sequence encoding restriction endonuclease recognition sequence --Forward primer, as well as --Reverse primer binding site, and The set of oligonucleotides includes a first oligonucleotide and a second oligonucleotide, which form an antiparallel double-stranded structure within the region of the restriction endonuclease recognition sequence when they are close to each other, thereby forming a double-stranded restriction site.

2. The composition of claim 1, wherein one or more of the oligonucleotides further comprises one or more of a modification coding region, a barcode region, a unique molecular identifier, and a fluorescent probe target.

3. The composition according to claim 1 or 2, wherein the body comprises a polyacrylamide material containing a pyrolytic element.

4. The composition according to claim 1 or 2, wherein the main body is configured to dissolve in a controlled environment.

5. The composition of claim 3, wherein the matrix is ​​configured to dissolve in a controlled environment.

6. The composition of claim 4, wherein the body further comprises a fluorescent tag that generates a fluorescent signal indicating the dissolution of the body.

7. The composition of claim 5, wherein the body further comprises a fluorescent tag that generates a fluorescent signal indicating the dissolution of the body.

8. The composition of claim 2, wherein the barcode area comprises two or more sets of barcode unit subsequences, wherein more than one of the two or more sets of barcode unit subsequences includes a common handle shared throughout a set of barcode unit subsequences during assembly.

9. The composition of claim 2, wherein the barcode area comprises two or more non-random sequences attached to a single subject.

10. The composition of claim 9, wherein the oligonucleotides comprise a first subset of oligonucleotides having a first barcode region, a second subset of oligonucleotides having a second barcode region, and a third subset of oligonucleotides having a third barcode region, the first barcode region having a first sequence, the second barcode region having a second sequence, and the third barcode region having a third sequence.

11. The composition of claim 2, wherein the oligonucleotide group comprises two or more barcode regions, said two or more barcode regions being linked to two or more polymerase chain reaction (PCR) handles or oligonucleotide binding regions.

12. The composition of claim 1 or 2, wherein one or more oligonucleotides of the group of oligonucleotides further comprise a cleavage region configured to separate at least a portion of the one or more oligonucleotides from the host.

13. The composition according to claim 12, wherein: (a) The cleavage region contains dU for the USER sequence; (b) The cleavage region contains a cleavable element located between a first fluorophore and a fluorescence quencher, the first fluorophore being located at a first end of the cleavable element and the fluorescence quencher being located at a second end of the cleavable element; (c) The cleavage region contains a cleavable element located between the body and the fluorophore; or (d) The cleavage region contains a thermally unstable site.

14. The composition according to claim 1 or 2, wherein the junction region comprises dendritic macromolecules.

15. The composition according to claim 1 or 2, wherein the body is configured to swell in response to a buffer solution transition.

16. The composition of claim 15, wherein the body is configured to be trapped within micropores in a swollen state.

17. The composition of claim 3, wherein the bulk is configured to dissolve in the presence of a reducing agent.

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